Use of cyclosporin alkyne analogues for preventing or treating viral-induced disorders

ABSTRACT

The present invention relates to methods of preventing or treating a mammal with a viral-induced disorder. The method involves administering to the mammal a therapeutically effective amount of a compound represented by Formula I, as shown below: 
                         
or a pharmaceutically acceptable salt thereof, with X, R 0 , and R 1  defined herein, under conditions effective to prevent or treat the viral-induced disorder.

FIELD OF THE INVENTION

The present invention discloses novel cyclosporin alkyne analogues andtheir utility as pharmaceutical agents for prevention and treatment ofviral-induced diseases. Methods for preparation of such compounds arealso disclosed.

BACKGROUND OF THE INVENTION

Cyclosporin A (CsA), a neutral cyclic undecapeptide isolated from thefungus Tolypocladium inflatum and currently marketed as NEORAL ® andSANDIMMUNE ® (Novartis, Basel, Switzerland), has been widely used forthe prevention of organ transplant rejection. The molecular basis forthe immunosuppressant activity of cyclosporin A and cyclosporinanalogues begins with the passive diffusion of the cyclosporin (Cs)molecule into the cell, followed by binding to its intracellularreceptor, cyclophilin A (CypA). CypA belongs to a family of proteinsthat catalyze cis-trans peptidyl-prolyl isomerization, i.e., PPIase, arate-limiting step in protein folding. CsA and other cyclosporinanalogues bind to the active site of CypA. However, immunosuppression isnot believed to be due to the inhibition of CypA PPIase activity. Thetarget of the CsA-CypA complex is a Ca²⁺-calmodulin-dependentserine-threonine-specific protein phosphatase, calcineurin. In T-cellsresponding to antigen presentation, an increase in intracellular Ca²⁺activates calcineurin, which subsequently dephosphorylates thetranscription factor called the nuclear factor of activated T-cells(“NFAT”). Dephosphorylated NFAT undergoes a molecular change, e.g.,homodimerization that allows it to cross into the nucleus, and promotesthe expression of T-cell activation genes. CsA and otherimmunosuppressive cyclosporin derivatives inhibit calcineurin whichresults in the inhibition of expression of cytokine genes, e.g.,interleukin-2 (IL-2) that promotes T-cell activation and proliferation,i.e., immunosuppressive activity.

Human Immunodeficiency Viruses and Cyclosporin A orNon-Immunosuppressive Cyclosporins

Human immunodeficiency viruses (“HIVs”) are lentiviruses, a family ofmammalian retroviruses evolved to establish chronic persistent infectionwith gradual onset of clinical symptoms. There are two major families ofHIV. Most of the epidemic involves HIV-1; HIV-2 is a close relativewhose distribution is concentrated in western Africa.

Human cyclophilins A and B have been identified as cellular proteinswhich bind specifically to HIV-1 Gag polyprotein, p555^(gag). Gagproteins play a major role in several steps of the virus life cycle,including the assembly and release of virions (Willis et al., “Form,Function, and Use of Retroviral Gag Proteins,” AIDS 5:639-654 (1991)). Acleavage product of the Gag polyprotein, the capsid protein, has beenshown to bind specifically to cyclophilin A. Cyclophilin A isfunctionally associated with the HIV-1 virions through interaction withthe Gag polyprotein. This interaction between cyclophilin A and Gagproteins is inhibited by the immunosuppressive drug, cyclosporin A(Thali et al., “Functional Association of Cyclophilin A With HIV-1Virions,” Nature 372:363-365 (1994)).

Cyclosporin A has demonstrated in vitro antiviral activity against HIV-1(Karpas et al., “Inhibition of Human Immunodeficiency Virus and Growthof Infected T-cells by the Immunosuppressive Drugs Cyclosporin A and FK506, ” Proc. Natl. Acad. Sci. USA 89:8351-8355 (1992)); however, initialin vivo studies in which cyclosporin A was administered as a monotherapyin HIV-infected patients at advanced stages of disease did not show abeneficial effect from the treatment (Levy et al., “Long-Term Follow-Upof HIV Positive Asymptomatic Patients Having Received Cyclosporin A,”Adv. Ex. Med. Biol. 374:229-234 (1995)). U.S. Pat. No. 4,814,323 toAndrieu et al. reported that administration of cyclosporins may be usedfor the prevention of AIDS in patients infected with the virus beforethe appearance of the AIDS symptoms, that is patients with no symptomsor patients with AIDS related complex.

Highly active antiretroviral therapy (“HAART”) has dramaticallydecreased the HIV-related morbidity and mortality rates amongHIV-infected patients and the transmission of HIV from mother to childby efficiently suppressing viral replication (Palella et al., “DecliningMorbidity and Mortality Among Patients With Advanced HumanImmunodeficiency Virus Infection,” N. Eng. J. Med. 338:853-860 (1998)).Limitations of HAART have become better understood. Thus, the virus canbe suppressed to undetectable levels but not eradicated. In addition,there is an ever-growing list of side effects, the eventual developmentof resistance, and the cost and complexity of HAART regimens that mustbe contended with.

HAART covers a broad range of antiretroviral agents that includenucleoside reverse transcriptase inhibitors (“NRTI”), nonnucleosidereverse transcriptase inhibitors (“NNRTI”), HIV protease inhibitors, andfusion inhibitors. Specific examples of antiviral agents from each ofthese families include: Zidovudine, Didanosine, Stavudine, andLamivudine from the NRTI antiviral class; Nevirapine, Efavirenz, andDelavirdine from the NNRTI antiviral class; Saquinovir, Indinavir, andRitonavir from the HIV protease inhibitor class; and Enfuvirtide fromthe fusion inhibitor antiviral class.

From an immunological standpoint, the introduction of HAART allows foronly a partial immune reconstitution. Indeed, ex vivo measures of immunefunction do not generally normalize and, most importantly, HIV-specificT cell responses remain almost invariably impaired. Though severalvariables have been identified that correlate with the degree of immunereconstitution during HAART, the actual underlying mechanism(s)responsible for such an incomplete immune reconstitution are stillpoorly understood and likely reflect the severe HIV-driven perturbationsin T cell dynamics and homeostasis and the interaction between host andviral factors (Douek, “Disrupting T-Cell Homeostasis: How HIV-1Infection Causes Disease,” AIDS Rev. 5:172-177 (2003)).

A strategy aimed at the broadest immune reconstitution, possiblyovercoming the limitations of HAART, consists in the adjuvant use ofimmunomodulants. By combining cyclosporin A with HAART, the goal is tocontain the immune activation, either virus-specific or owing tonon-specific “by-stander” activation. Results from pilot studies inHIV-infected patients has shown that the rapid shutdown of T-cellactivation induced by cyclosporin A has produced a more rapid and stableincrease in CD4+ T-cells and a significant long-term increase in IFN-γsecreting CD4+ and CD4+CCR7− T-cells, establishing a more favorableimmunological set-point (Bandera et al., “Immunomodulants in HIVInfection,” Expert Opin. Ther. Patents 15(9): 1115-1131 (2005)).Determination of the long-term efficacy must be assessed in order tounderstand if this approach truly has value.

SDZ NIM 811 is a cyclosporin analogue that is completely devoid ofimmunosuppressive activity but exhibits potent and selective anti-HIV-1activity (Mlynar et al., “The Non-Immunosuppressive Cyclosporin AAnalogue SDZ NIM 811 Inhibits Cyclophilin A Incorporation Into Virionsand Virus Replication in Human Immunodeficiency Virus Type-1-InfectedPrimary and Growth-Arrested Cells,” J. General Virology 78:825-835(1997)). SDZ NIM 811 does not prevent the activation of CD4+ T-cellactivation as cyclosporin A does. In a manner similar to cyclosporin A,it is proposed that SDZ NIM 811 interferes with the HIV-1Gag-cyclophilin A interaction to effect its antiviral activity.

SDZ NIM 811 does not inhibit calcineurin and possesses none of theimmunosuppressive activity of cyclosporin A. The potent inhibition ofcalcineurin by cyclosporin, in addition to being responsible for thepotent immunosuppressive activity of cyclosporin A, is also believed tobe the cause of the toxicity and the narrow therapeutic index of thisdrug. Separation of immunosuppressive and antiviral activity could leadto novel antiviral cyclosporins with fewer side effects and improvedtherapeutic index. Elucidation of structure activity relationships forcyclosporins permits the design of non-immunosuppressive cyclosporinderivatives that retain potent (cyclophilin A) PPIase activity toachieve this goal (Bartz et al., “Inhibition of Human ImmunodeficiencyVirus Replication by Non-Immunosuppressive Analogs of Cyclosporin A,”Proc. Natl. Acad. Sci. USA 92:5381-5385 (1995)). European Patent No. 484281, U.S. Pat. No. 5,767,069, U.S. Pat. No. 5,948,884, and French PatentNos. 2,757,520, 2,757,521, and 2,757,522 disclose non-immunosuppressivecyclosporins with antiviral activity.

Hepatitis C Virus and Cyclosporin A

Recently, cyclosporin A, the most widely prescribed immunosuppressivedrug, was reported to be clinically effective against hepatitis C viral(HCV) infection (Nakagawa et al., “Specific Inhibition of Hepatitis CVirus Replication by Cyclosporin A,” Biochem. Biophys. Res. Commun.313:42-47 (2004)). The authors of the Nakagawa et al. paper state thatcertain chaperone activities, such as those of cyclophilins, may becrucial for the processing and maturation of the viral proteins and forviral replication.

A subsequent controlled clinical trial showed that a combination ofcyclosporin A with interferon α2b is more effective than interferonmonotherapy, especially in patients with high viral loads (Inoue et al.,“Combined Interferon α2b and Cyclosporin A in the Treatment of ChronicHepatitis C: Controlled Trial,” J. Gastroenterol. 38:567-572 (2003)).

PCT International Patent Publication No. WO 2006/005610 recentlydescribed the use of a combination of cyclosporin A and pegylatedinterferon for treating hepatitis C viral infection. In addition, PCTInternational Patent Publication No. WO 2005/021028 relates to the useof non-immunosuppressive cyclosporins for treatment of HCV disorders.Also, Paeshuyse et al., “Potent and Selective Inhibition of Hepatitis CVirus Replication by the Non-Immunosuppressive Cyclosporin AnalogueDEBIO-025, ” Antiviral Research 65(3):A41 (2005) recently publishedresults for a non-immunosuppressive cyclosporin analogue, DEBIO-025,that exhibited potent and selective inhibition of hepatitis C virusreplication. Notably, the cyclosporin derivative DEBIO-025 is alsoeffective for the treatment of HIV-1 (Rosenwirth et al., “Debio-025, ANovel Non-Immunosuppressive Cyclosporine Analog with Potent Anti-HumanImmunodeficiency Virus Type 1 Activity: Pharmacological Properties andMode of Action,” Antiviral Research 65(3):A42-A43 (2005)). Debio-025does possess potent binding affinity for cyclophilin A.

There is still a large need for novel cyclosporin analogues that havetherapeutic utility in the treatment of viral-induced diseases.

The present invention is directed to achieving these objectives.

SUMMARY OF THE INVENTION

The present invention relates to a method of preventing or treating amammal with a viral-induced disorder. The method involves administeringto the mammal a therapeutically effective amount of a compound havingthe following formula:

where:

-   X is OH or OAc;-   R₀ is H, CH₂OH, or CH₂OR₂;-   R₁ is selected from the group consisting of:    -   hydrogen;    -   halogen;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain containing substitution or substitutions selected from the        group consisting of deuterium, halogen, nitrogen, sulfur, and        silicon atom or atoms;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain containing a function group or function groups selected        from the group consisting of alcohol, ether, aldehyde, ketone,        carboxylic ester, and amide;    -   C₂-C₄ saturated or unsaturated, straight or branched carbon        chain containing an aryl or a heteroaryl;    -   C₃-C₆-substituted and unsubstituted cycloalkyl;    -   substituted and unsubstituted aryl;    -   substituted and unsubstituted heteroaryl;    -   —CH₂OH;    -   —CHO;    -   —CH═N—OR₃; and    -   —CH═N—NR₃R₄;-   R₂ is selected from the group consisting of:    -   alkanoyl;    -   alkenoyl;    -   alkynoyl;    -   aryloyl;    -   arylalkanoyl;    -   alkylaminocarbonyl;    -   arylaminocarbonyl;    -   arylalkylaminocarbonyl;    -   alkyloxycarbonyl;    -   aryloxycarbonyl; and    -   arylalkyloxycarbonyl;-   R₃ or R₄ are the same or different and independently selected from    the group consisting of:    -   hydrogen;    -   C₁-C₆ saturated straight or branched carbon chain;    -   C₃-C₆ unsaturated straight or branched carbon chain;    -   C₃-C₆-substituted and unsubstituted cycloalkyl;    -   C₁-C₄ carbon chain containing an aryl or heteroaryl;    -   substituted and unsubstituted aryl;    -   substituted and unsubstituted heteroaryl;    -   alkanoyl;    -   alkenoyl;    -   alkynoyl;    -   aryloyl;    -   arylalkanoyl;    -   alkylaminocarbonyl;    -   arylaminocarbonyl;    -   arylalkylaminocarbonyl;    -   alkyloxycarbonyl;    -   aryloxycarbonyl; and    -   arylalkyloxycarbonyl; and-   R₃ together with R₄ results in the formation of a cyclic moiety of    C₂-C₆ optionally containing heteroatom or heteroatoms,    or a pharmaceutically acceptable salt thereof,    under conditions effective to prevent or treat the viral-induced    disorder.

The present invention discloses chemically modified cyclosporinanalogues containing a carbon-carbon triple bond on the side chain ofthe position one amino acid and optionally a substitution on theposition three amino acid of cyclosporin A. In particular, the presentinvention discloses novel cyclosporin alkyne analogues containing aconjugated system of a carbon-carbon triple bond with an aryl, acarbon-carbon double bond, a carbon-nitrogen double bond, or anothercarbon-carbon triple bond.

The present invention discloses novel cyclosporin analogues which areeffective as antiviral agents. The cyclosporin derivatives of thepresent invention used to treat viral infections may possess potentimmunosuppressive activity (via inhibition of calcineurin) or may becompletely devoid of immunosuppressive activity (do not inhibitcalcineurin). However, the mechanism that the immunosuppressive andnon-immunosuppressive cyclosporin compounds share is their activity atcyclophilin A.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the results from a concanavalin A (ConA)-stimulatedmurine splenocyte assay, where the novel cyclosporin analogue compoundsof the present invention (disclosed in Examples 25 and 10) are shown topossess enhanced or similar potency in immunosuppression, compared tocyclosporin A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of preventing or treating amammal with a viral-induced disorder. The method involves administeringto the mammal a therapeutically effective amount of a compound havingthe following formula:

where:

-   X is OH or OAc;-   R₀ is H, CH₂OH, or CH₂OR₂;-   R₁ is selected from the group consisting of:    -   hydrogen;    -   halogen;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain containing substitution or substitutions selected from the        group consisting of deuterium, halogen, nitrogen, sulfur, and        silicon atom or atoms;    -   C₂-C₆ saturated or unsaturated, straight or branched carbon        chain containing a function group or function groups selected        from the group consisting of alcohol, ether, aldehyde, ketone,        carboxylic ester, and amide;    -   C₂-C₄ saturated or unsaturated, straight or branched carbon        chain containing an aryl or a heteroaryl;    -   C₃-C₆-substituted and unsubstituted cycloalkyl;    -   substituted and unsubstituted aryl;    -   substituted and unsubstituted heteroaryl;    -   —CH₂OH;    -   —CHO;    -   —CH═N—OR₃; and    -   —CH═N—NR₃R₄;-   R₂ is selected from the group consisting of:    -   alkanoyl;    -   alkenoyl;    -   alkynoyl;    -   aryloyl;    -   arylalkanoyl;    -   alkylaminocarbonyl;    -   arylaminocarbonyl;    -   arylalkylaminocarbonyl;    -   alkyloxycarbonyl;    -   aryloxycarbonyl; and    -   arylalkyloxycarbonyl;-   R₃ or R₄ are the same or different and independently selected from    the group consisting of:    -   hydrogen;    -   C₁-C₆ saturated straight or branched carbon chain;    -   C₃-C₆ unsaturated straight or branched carbon chain;    -   C₃-C₆-substituted and unsubstituted cycloalkyl;    -   C₁-C₄ carbon chain containing an aryl or heteroaryl;    -   substituted and unsubstituted aryl;    -   substituted and unsubstituted heteroaryl;    -   alkanoyl;    -   alkenoyl;    -   alkynoyl;    -   aryloyl;    -   arylalkanoyl;    -   alkylaminocarbonyl;    -   arylaminocarbonyl;    -   arylalkylaminocarbonyl;    -   alkyloxycarbonyl;    -   aryloxycarbonyl; and    -   arylalkyloxycarbonyl; and-   R₃ together with R₄ results in the formation of a cyclic moiety of    C₂-C₆ optionally containing heteroatom or heteroatoms,    or a pharmaceutically acceptable salt thereof,    under conditions effective to prevent or treat the viral-induced    disorder.

One embodiment of the present invention is the above compound of FormulaI, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ is H.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of F, Cl, Br, and I.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of CH═CH₂, CH═CHCH₃, CH═CHCH₂CH₃,C(CH₃)═CH₂, CH═CD₂, CH═CHCD₃, and CH═CDCD₃, and where the carbon-carbondouble bond is a cis or a trans geometric isomer or a mixture of bothcis and trans geometric isomers.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of CH═CHF, CH═CHCl, CH═CHBr, CH═CHI,CH═CF₂, and CH═CCl₂, and where the carbon-carbon double bond is a cis ora trans geometric isomer or a mixture of both cis and trans geometricisomers.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of C≡CH, C≡CCH₃, C≡CCD₃, C≡CCH₂CH₃,C≡CCH₂CH₂CH₃, and C≡C-cyclopropyl.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of CH₂C≡CH, CH₂C≡CCH₃, CH₂C≡CCH₂CH₃,CH₂CH═CH₂, CH₂CH═CHCH₃, and CH₂CH═CHCH₂CH₃ and where the carbon-carbondouble bond is a cis or a trans geometric isomer or a mixture of bothcis and trans geometric isomers

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of C≡C—C≡CH, C≡C—C≡CCH₃, C≡CCH═CH₂,C≡CCH═CHCH₃, CH═CHC≡CH, CH═CHC≡CCH₃, CH═CHCH═CH₂, and CH═CHCH═CHCH₃ andwhere the carbon-carbon double bond is a cis or a trans geometric isomeror a mixture of both cis and trans geometric isomers

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ iscyclopropyl.

Another embodiment of the present invention is the above compound ofFormula I, where: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OAc; and R₁ isselected from the group consisting of CH₂OH, —CHO, CH(OH)CH₃, C(═O)CH₃,CH═N—OCH₃, CH═N—OCH₂CH₃, CH═N—NHCH₃, and CH═N—N(CH₃)₂.

Other embodiments of the present invention include the above compound ofFormula I, where: X=OH or OAc; R₀=H; and R₁ is selected from the groupconsisting of H, C₆H₅—, p-FC₆H₄—, p-CH₃OC₆H₄—, 2-thiophenyl, CH₂Ph,CH₂CH═CH₂, CH₂C≡CH, CH₂C≡CHCH₃, CH₂C≡CHSi(CH₃)₃, Br, CH₂Cl, CH═CH₂,CH═CHCH₃(trans), CH═CHCH₃(cis), CH═CHCl(trans), CH═CHCl(cis),CH═CHSi(CH₃)₃(trans), C(CH₃)═CH₂, CH═CHPh, CH═CHCO₂Et(cis), CH═C═CH₂,C≡CH, C≡CCH₃, C≡CCD₃, C≡CCH₂CH₃, C≡CC₄H₉, C≡CSi(CH₃)₃, C≡C-3-thiophene,C≡C-Ph, C≡CBr, C≡C-cyclopropyl, C≡C-cyclohexyl, C≡CCH₂OH, C≡CCH₂OCH₃,C≡CCH₂SCH₂CH₃, C≡CCH₂N(CH₃)₂, C≡CCH═CH₂, C≡CC(CH₃)═CH₂,C≡CCH═CHCH₃(cis), C≡CCH═CHCH₃(trans), CH═CHC≡CH, CH═CHC≡CCH₂CH₂CH₃,CH═CHC≡C-cyclopropyl, CH₂OH, CHO, C═N—OCH₃, and C═N—N(CH₃)₂.

Other embodiments of the present invention include the above compound ofFormula I, where: X=OH or OAc; R₀=CH₂OH or CH₂OAc; and R₁ is selectedfrom the group consisting of H, CH═CH₂, CH═CHCH₃(cis), CH═CHCH₃ (trans),and CH═CHCl(cis).

In particular, the present invention relates to novel cyclosporinanalogues containing a carbon-carbon triple bond on the side chain ofthe position one amino acid and optionally a substitution on theposition three amino acid of cyclosporin A. More particularly, thepresent invention relates to novel cyclosporin alkyne analogues, inwhich the carbon-carbon triple bond conjugating with an aryl, or aheteroaryl, or a carbon-carbon double bond, or a carbon-nitrogen doublebond, or another carbon-carbon triple bond is incorporated.

A carbon-carbon triple bond exists in many natural products (Gung etal., “Total Synthesis of (S)-(−)-(E)-15,16-Dihydrominquartynoic Acid: AHighly Potent Anticancer Agent,” J. Org. Chem., 69:3488-3492 (2004); Itoet al., “Cytotoxic Polyacetylenes from the Twigs of Ochanostachysamentacea,” J. Nat. Prod., 64:246-248 (2001), which are herebyincorporated by reference in their entirety). It is well known to usealkynes as pharmaceutical agents. However, only one cyclosporin alkyne,in which a carbon-carbon triple bond replaces the carbon-carbon doublebond on the side-chain of the position one amino acid of cyclosporin A,is known in the literature. Unfortunately, this modificationsignificantly reduces the immunosuppressive activity of cyclosporin A,where this known cyclosporin alkyne shows only 10% relativeimmunosuppressive activity, compared to cyclosporin A (Rich et al.,“Synthesis, Conformation, and Immunosuppressive Activities of ThreeAnalogues of Cyclosporin A Modified in the 1-Position,” J. Med. Chem.,33:999-1009 (1990), which is hereby incorporated by reference in itsentirety). In contrast, the novel cyclosporin alkyne analogues of thepresent invention, which contain a conjugated system of a carbon-carbontriple bond and a carbon-carbon double bond or a carbon-carbon triplebond, possess enhanced immunosuppressive activity over cyclosporin A.

The present invention also discloses methods for preparing compoundsrepresented by Formula I.

The starting material for the preparation of the compounds of thepresent invention is cyclosporin A. The structure of cyclosporin A, acycloundecapeptide, and the position numbering for each amino acid inthe ring is shown below:

Cyclosporin A can also be represented by Formula IIa, as shown below:

The novel cyclosporin analogues of the present invention are derivedfrom cyclosporin A or cyclosporin diol (Formula IIb), a key intermediateprepared by modification on the position three amino acid of cyclosporinA. As shown in Scheme 1, the cyclosporin diol intermediate can beprepared by deprotonation of cyclosporin A with lithium diisopropylamide(LDA) followed by treatment with formaldehyde (Seebach et al,“Modification of Cyclosporin A: Generation of an Enolate at theSarcosine Residue and Reaction with Electrophiles,” Helv. Chim. Acta,76:1564-1590 (1993), which is hereby incorporated by reference in itsentirety).

According to one embodiment of the present invention, novel cyclosporinanalogues can be prepared by replacing the carbon-carbon double bond onthe side chain of the position one amino acid of cyclosporin A with acarbon-carbon triple bond. As depicted in Scheme 2, acetylation ofcyclosporin A (Formula IIa) or the cyclosporin diol intermediate ofFormula IIb with acetic anhydride, followed by oxidative cleavage of thedouble bond with ozone, generates the cyclosporin aldehyde of FormulaIII smoothly. Treatment of the cyclosporin aldehyde of Formula III withdimethyl (1-diazo-2-oxopropyl)phosphonate in the presence of potassiumcarbonate in methanol provides cyclosporin alkyne (Formula I, X=OH) ingood yield (Müller et al, An Improved One-Pot Procedure for theSynthesis of Alkynes from Aldehydes,” Synlett, 521-522 (1996), which ishereby incorporated by reference in its entirety). The acetyl protectinggroup can be removed under these reaction conditions to give the freealcohol directly.

The cyclosporin aldehyde of Formula III can also be converted to thecyclosporin alkyne of Formula I (X=OH or OAc) via an alternate method(Scheme 2). Treatment of cyclosporin aldehyde withlithiotrimethylsilyldiazomethane affords the cyclosporin alkyne ofFormula I (X=OH, R₀=H or CH₂OH) in good yield (Ohira et al, “Generationof Alkylidenecarbenes by the Alkenation of Carbonyl Compounds withLithiotrimethylsilyldiazomethane,” J. Chem. Soc. Chem. Commun., 721-722(1992), which is hereby incorporated by reference in its entirety),while the reaction of cyclosporin aldehyde withlithiotrimethylsilyldiazomethane, followed by acidic workup (Ac₂O),provides the acetyl cyclosporin alkyne of Formula I (X=OAc, R₀=H orCH₂OAc).

Using the above described cyclosporin alkyne (Formula I, X=OH) as a keyintermediate, many novel cyclosporin alkyne derivatives can be preparedvia palladium or nickel-mediated couplings. As shown in Scheme 3,Sonogashira coupling of cyclosporin alkyne (Formula I) with various arylhalides, heteroaryl halides, and vinyl halides provides novelcyclosporin arylated alkynes of Formula IV and cyclosporin yne-eneanalogues of Formula V, respectively. Similarly, the application ofpalladium-catalyzed coupling to the same key intermediate, cyclosporinalkyne (Formula I), with alkynyl halides leads to the preparation ofnovel cyclosporin diynes of Formula VI. Utilizing this method, acarbon-carbon triple bond could be introduced step by step to provide aconjugated system of multiple carbon-carbon triple bonds, such astriynes and tetraynes.

As shown in Scheme 4, the cyclosporin diynes of Formula VI can beprepared using an alternative approach. Bromination of cyclosporinalkyne (Formula I, X=OAc, R₁=H) with N-bromosuccinimide in the presenceof silver nitrate affords cyclosporin alkynyl bromide (Formula VIII).Using this method, other cyclosporin alkynyl halides, such ascyclosporin alkynyl iodide, can be obtained with N-iodosuccinimideinstead of N-bromosuccinimide. Palladium-catalyzed coupling ofcyclosporin alkynyl bromide (or cyclosporin alkynyl iodide) with variousalkynes affords cyclosporin diynes of Formula VI smoothly.

Another embodiment of the present invention relates to the alkylation ofcyclosporine alkyne (Formula I, R₁=H). As shown in Scheme 5, thetreatment of cyclosporine alkyne (Formula I, R₁=H) with alkyl halides oraldehyde in the presence of a base (cesium carbonate,benzyltrimethylammonium hydroxide, or other strong bases) provides thealkylated cyclosporin alkyne (Formula IX).

Another embodiment of the present invention relates to the incorporationof a carbon-nitrogen double bond (C═N) in the cyclosporin alkyne ofFormula I. As shown in Scheme 6, the reaction of cyclosporin alkyne(Formula I, R₁=H) with formaldehyde, using benzyltrimethylammoniumhydroxide as a base, provides the cyclosporin diol of Formula X.Selective protection of the primary alcohol of the cyclosporin diol withtert-butyldimethylsilyl choloride, followed by acetylation of the secondalcohol with acetic anhydride and then desilylation withtetrabutylammonium fluoride, affords the mono-alcohol (Formula XI)smoothly. Swern oxidation of the mono-alcohol affords the cyclosporinaldehyde of Formula XII. Treatment of the aldehyde with hydroxylamine,alkyloxyamines (RONH₂), or hydrazines (R₂NNH₂) affords the correspondingcyclosporin oximes (CH═N—OR) and hydrazones (CH═N—NR₂) of Formula VII,respectively.

Some of the compounds disclosed in the present invention are useful asimmunosuppressive agents. Administration of these compounds suppressesthe immune response in organ transplant patients and, thus, preventsallograft rejection. The compounds of the present invention possessenhanced or similar immunosuppressive activity, compared to cyclosporinA. For example, as shown in FIG. 1, the cyclosporin alkyne analoguecompound disclosed in Example 25 demonstrates immunosuppressive activitytwo times more potent over cyclosporin A, while the cyclosporin alkyneanalogue compound disclosed in Example 10 shows similar potency tocyclosporin A in the concanavalin A (ConA) stimulated murine splenocyteassay. Table 1 shows the immunosuppressive activities of several novelcyclosporin alkyne analogue compounds disclosed in the presentapplication. (The third column in Table 1 contains cyclosporin Apositive control values included for comparison.)

TABLE 1 Immunosuppressive Activities of Novel Cyclosporin AlkyneAnalogue Compounds of the Present Invention Example Where the NovelCyclosporin Alkyne Analogue Compound is Disclosed IC₅₀ (ng/mL) IC₅₀(ng/mL) of CsA Example 7 25 15 Example 10 25 20 Example 13 42 15 Example14 32 15 Example 22 15 18 Example 25 12 20 Example 27 12 31 Example 2920 31 Example 33 43 15 Example 35 77 18 Example 38 24 18 Example 43 4618

The compounds disclosed in the present invention are useful for theprevention or treatment of viral-induced disorders that are dependentupon the presence of cyclophilin A. The compounds of the presentinvention used to treat these viral infections may possess potentimmunosuppressive activity (via inhibition of calcineurin) or may becompletely devoid of immunosuppressive activity (do not inhibitcalcineurin). However, the mechanism that the immunosuppressive andnon-immunosuppressive cyclosporin compounds share is their activity atcyclophilin A.

Cyclophilin A enzyme activity, i.e., peptidyl-prolyl cis-trans isomeraseactivity, is important to the folding and trafficking of proteins. TheHIV infectivity of CD4+ T-cells and viral replication are dependent uponthe incorporation of cyclophilin A into HIV-1 virions throughinteractions with the Gag polyprotein. Inhibition of the cyclophilin Aenzyme activity is necessary and sufficient for anti-HIV-1 activity.

In one embodiment of the present invention, the viral-induced disorderis a human immunodeficiency virus (HIV)-induced disorder. Thus,compounds of the present invention that lack immunosuppressant activityas determined by the Concanavalin A (Con A)-stimulated murine splenocyteassay but retain potent peptidyl prolyl isomerase (PPIase) inhibitory(cyclophilin A) activity may possess anti-HIV activity. In addition,compounds of the present invention that have immunosuppressive activityas determined by the Con A-stimulated murine splenocyte assay and alsopossess potent PPIase inhibitory (cyclophilin A) activity may possessanti-HIV activity.

In vitro biological assays that allow the determination of bindingaffinity to cyclophilin A or allow the determination of inhibition ofpeptidyl cis-trans isomerase activity are described in Handschumacher etal., “Cyclophilin: A Specific Cytosolic Binding Protein for CyclosporinA,” Science 226:544-547 (1984) and Kofron et al., “Determination ofKinetic Constants for Peptidyl Prolyl Cis-Trans Isomerases by anImproved Spectrophotometric Assay,” Biochemistry 30:6127-6134 (1991),respectively, which are both hereby incorporated by reference in theirentirety.

The in vitro anti-HIV activity of compounds of the present invention canbe measured in established cell line cultures as described by Mayaux etal., “Triterpene Derivatives That Block Entry of Human ImmunodeficiencyVirus Type 1 Into Cells,” Proc. Natl. Acad. Sci. USA 91:3564-3568(1994), which is hereby incorporated by reference in its entirety.

In another embodiment of the present invention, the compound of thepresent invention is administered in combination with antiretroviralagents, such as nucleoside reverse transcriptase inhibitors,nonnucleoside reverse transcriptase inhibitors, human immunodeficiencyvirus protease inhibitors, fusion inhibitors, and combinations thereof.Examples of nucleoside reverse transcriptase inhibitors include, but arenot limited to, Zidovudine, Didanosine, Stavudine, and Lamivudine.Examples of nonnucleoside reverse transcriptase inhibitors include, butare not limited to, Nevirapine, Efavirenz, and Delavirdine. Examples ofhuman immunodeficiency virus protease inhibitors include, but are notlimited to, Saquinovir, Indinavir, and Ritonavir. Examples of fusioninhibitors include, but are not limited to, Enfuvirtide.

Although cyclophilin PPIase activity would appear to be implicated inanti-HCV activity as it is for anti-HIV-1 activity, the hepatitis Cvirus (HCV) proteins that may interact with cyclophilin A have yet to beidentified. In another embodiment of the present invention, theviral-induced disorder is a HCV-induced disorder. Hepatitis C infectionsor HCV induced disorders are, for example, chronic hepatitis, livercirrhosis, or liver cancer (e.g., hepatocellular carcinoma). Thus,compounds of the present invention that lack immunosuppressant activityas determined by the Concanavalin A (Con A)-stimulated murine splenocyteassay but retain potent peptidyl prolyl isomerase (PPIase) inhibitory(cyclophilin A) activity may possess anti-HCV activity. In addition,compounds of the present invention that have immunosuppressive activityas determined by the Con A-stimulated murine splenocyte assay and alsopossess potent PPIase inhibitory (cyclophilin A) activity may possessanti-HCV activity. The compounds of the present invention may also beused as a prophylactic treatment for neonates born to HCV-infectedmothers, for healthcare workers exposed to the virus, or for transplantrecipients, e.g., organ or tissue transplant (e.g. liver transplant)recipients, to eliminate possible recurrent infection aftertransplantation.

In another embodiment of the present invention, the compound of thepresent invention is administered in combination with an interferon.Examples of interferons include, but are not limited to, interferon α2aand interferon α2b. The interferon can be a pegylated interferon.Examples of interferons include, but are not limited to, pegylatedinterferon α2a or pegylated interferon α2b.

Utility of the immunosuppressive or non-immunosuppressive cyclosporincompounds of the present invention in treating diseases or conditionsfrom HCV infection can be demonstrated in standard animal or clinicaltests in accordance with the methods described in Examples 54 and 55,for example.

Some of the compounds disclosed in the present invention also possessutility in the treatment of autoimmune and chronic inflammatory diseasessuch as asthma, rheumatoid arthritis, multiple sclerosis, psoriasis, andulcerative colitis, to name only a few.

The compounds disclosed in the present invention are also useful for thetreatment of ocular allergy and dry eye. Allergan is currently marketinga topical formulation of cyclosporin A called RESTASIS™ (cyclosporinophthalmic emulsion) for the treatment of keratoconjunctivitis sicca orchronic dry eye syndrome in patients whose tear production is presumedto be suppressed due to ocular inflammation. While the exact mechanismof RESTASIS™ is unknown, it is thought to act as an immunomodulator withanti-inflammatory effects (“Annual Update 2003: Ophthalmic Drugs” Drugsof the Future, 28(3): 287-307 (2003); Clark et al., “Ophthalmic DrugDiscovery,” Nature Reviews in Drug Discovery, 2:448-459 (2003), whichare hereby incorporated by reference in their entirety).

For treatment of the above mentioned diseases, therapeutically effectivedoses of the compounds of the present invention may be administeredorally, topically, parenterally, by inhalation spray, or rectally indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants, and vehicles. The termparenteral, as used herein, includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques.

The pharmaceutical compositions containing the active ingredient may bein the form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Thepharmaceutical compositions of the present invention contain the activeingredient formulated with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutical acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material, or formulation auxiliary of any type. Someexamples of pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch or potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil, and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; non-toxic, compatible lubricants such assodium lauryl sulfate and magnesium stearate; as well as coloringagents, releasing agents, sweetening, and flavoring and perfumingagents. Preservatives and antioxidants, such as ethyl or n-propylp-hydroxybenzoate, can also be included in the pharmaceuticalcompositions.

Dosage forms for topical or transdermal administration of compoundsdisclosed in the present invention include ointments, pastes, creams,lotions, gels, plasters, cataplasms, powders, solutions, sprays,inhalants, or patches. The active component is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers, as may be required. The ointments, pastes,creams and gels may contain, in addition to an active compound of thepresent invention, excipients such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

For nasal administration, the compounds disclosed in the presentinvention can be administered, as suitable, in liquid or powdered formfrom a nasal applicator. Forms suitable for ophthalmic use will includelotions, tinctures, gels, ointment and ophthalmic inserts, as known inthe art. For rectal administration (topical therapy of the colon), thecompounds of the present invention may be administered in suppository orenema form, in solution in particular, for Example in vegetable oil orin an oily system for use as a retention enema.

The compounds disclosed in the present invention may be delivered to thelungs by the inhaled route either in nebulizer form or as a dry powder.The advantage of the inhaled route, over the systemic route, in thetreatment of asthma and other diseases of airflow obstruction and/orchronic sinusitis, is that patients are exposed to very small quantitiesof the drug and the compound is delivered directly to the site ofaction.

Dosages of the compounds of the present invention employed for thetreatment of the maladies identified in the present invention will varydepending on the site of treatment, the particular condition to betreated, the severity of the condition, the subject to be treated (whomay vary in body weight, age, general health, sex, and other factors) aswell as the effect desired.

Dosage levels ranging from about 0.05 mg to about 50 mg per kilogram ofbody weight per day are useful for the treatment of the conditions ordiseases identified in the present invention. This means the amount ofthe compound disclosed in the present invention that is administeredwill range from 2.5 mg to about 2.5 gm per patient per day.

The amount of active ingredient that may be combined with thepharmaceutical carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. For example, a formulation intended for the oraladministration of humans may contain from 2.5 mg to 2.5 gm of activecompound of the present invention compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 to 95percent of the total composition. Dosage unit forms will generallycontain between from about 5 mg to about 500 mg of active compound ofthe present invention. Dosage for topical preparation will, in generalbe less (one tenth to one hundredth) of the dose required for an oralpreparation.

EXAMPLES Example 1 Preparation of Cyclosporin Acetate

A solution of cyclosporin A (5.0 g, 4.16 mmol), acetic anhydride (7.80mL, 83.2 mmol), and DMAP (760 mg, 6.2 mmol) in methylene chloride (40mL) was stirred overnight at room temperature under N₂ atmosphere.Saturated sodium bicarbonate solution (200 mL) was added to the solutionand stirred for an additional 2 h. The mixture was extracted with ether,washed with 1 N HCl, neutralized with saturated sodium bicarbonatesolution, washed with brine, dried over sodium sulfate, and concentratedin vacuo to afford cyclosporin acetate (4.92 g, 95%) as a white solid:¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J=9.6 Hz, 1H), 8.04 (d, J=6.9 Hz,1H), 7.51 (d, J=9.4 Hz, 1H), 7.47 (d, J=7.8 Hz, 1H), 5.67 (dd, J=11.0,4.0 Hz, 1H), 5.60-5.44 (m, 2H), 5.39 (dd, J=11.7, 3.7 Hz, 1H), 5.32-5.13(m, 4H), 5.06-4.93 (m, 2H), 4.85 (t, J=7.2 Hz, 1H), 4.77 (t, J=9.6 Hz,1H), 4.65 (d, J=13.7 Hz, 1H), 4.41 (t, J=7.0 Hz, 1H), 3.46 (s, 3H), 3.26(s, 3H), 3.24 (s, 3H), 3.21 (s, 3H), 3.10 (s, 3H), 2.68 (s, 3H), 2.66(s, 3H), 2.50-2.35 (m, 1H), 2.25-1.80 (m, 6H), 2.08 (s, 3H), 2.01 (s,3H), 1.75-1.55 (m, 6H), 1.45-0.75 (m, 55H); ESI MS m/z 1245[C₆₄H₁₁₃N₁₁O₁₃+H]⁺.

Example 2 Preparation of Acetyl Cyclosporin Aldehyde

Ozone was bubbled into a solution of cyclosporin acetate from Example 1(3.0 g, 2.4 mmol) in methylene chloride (70 mL) at −78° C. until a bluecolor was developed. The mixture was degassed with nitrogen for a fewmin and dimethylsulfide (3 mL) was added at −78° C. The reaction mixturewas allowed to warm to room temperature and stirred for 3 h. Thereaction mixture was concentrated in vacuo and the residue was dissolvedin ethyl acetate (300 mL), washed with water (2×70 mL) and brine (70mL), dried over sodium sulfate, filtered, and concentrated in vacuo toafford acetyl cyclosporin aldehyde (2.79 g, 94%) as a white solid. Thecrude product was carried to the next step without further purification:¹H NMR (300 MHz, CDCl₃) δ 9.60 (d, J=3.5 Hz, 1H), 8.55 (d, J=9.7 Hz,1H), 7.96 (d, J=6.8 Hz, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.46 (d, J=9.0 Hz,1H), 5.67 (dd, J=11.0, 3.8 Hz, 1H), 5.60-5.45 (m, 2H), 5.32 (dd, J=12.1,3.3 Hz, 1H), 5.24-5.10 (m, 2H), 5.08-4.90 (m, 2H), 4.84 (t, J=7.1 Hz,1H), 4.73 (t, J=9.6 Hz, 1H), 4.64 (d, J=13.8 Hz, 1H), 4.41 (t, J=7.0 Hz,1H), 3.46 (s, 3H), 3.29 (s, 6H), 3.21 (s, 3H), 3.08 (s, 3H), 2.67 (s,3H), 2.65 (s, 3H), 2.50-2.35 (m, 2H), 2.25-1.80 (m, 6H), 1.99 (s, 3H),1.75-1.55 (m, 3H), 1.50-0.75 (m, 57H); ESI MS m/z 1233[C₆₂H₁₀₉N₁₁O₁₄+H]⁺.

Example 3 Preparation of Cyclosporin Alkyne

To a stirred solution of acetyl cyclosporin aldehyde from Example 2(1.94 g, 1.57 mmol) in methanol (20 mL) was added a solution of dimethyl(1-diazo-2-oxopropyl)phosphonate (3.01 g, 15.7 mmol) in methanol (10 mL)followed by potassium carbonate (2.17 g, 15.7 mmol). The resulting greensuspension was stirred at room temperature overnight. The solution wasfiltered through diatomaceous earth and the filtrate was concentrated.The residue was dissolved in ethyl acetate (300 mL) and washed withwater (2×100 mL). The combined aqueous layers were extracted with ethylacetate (100 mL). The combined organic layers were washed with brine,dried over sodium sulfate, and concentrated to dryness. Purification bysemi-preparative HPLC gave cyclosporin alkyne (848 mg, 45%) as a whitesolid: ¹H NMR (300 MHz, CDCl₃) δ 7.86 (d, J=9.7 Hz, 1H), 7.62 (d, J=9.3Hz, 1H), 7.56 (d, J=6.9 Hz, 1H), 7.34 (d, J=7.7 Hz, 1H), 5.73-5.68 (m,1H), 5.57-5.45 (m, 2H), 5.22-4.45 (m, 12H), 4.03-3.98 (m, 1H), 3.49 (s,3H), 3.38 (s, 3H), 3.24 (s, 3H), 3.09 (s, 3H), 3.08 (s, 3H), 2.72 (s,3H), 2.70 (s, 3H), 2.50-0.64 (m, 66H); ESI MS m/z 1187[C₆₁H₁₀₇N₁₁O₁₂+H]⁺.

Example 4 Preparation of Cyclosporin Alkyne

To a −78° C. solution of (trimethylsilyl)diazomethane (4.6 mL, 2.0 Msolution in Et₂O, 9.2 mmol) in THF (5 mL) was added n-BuLi (3.4 mL, 2.5M solution in hexanes, 8.4 mmol) dropwise. The resulting yellowsuspension was stirred for 30 min, and then a solution of acetylcyclosporine aldehyde from Example 2 (1.03 g, 0.84 mmol) in THF (5 mL)was added dropwise. The mixture was stirred at −78° C. for 30 min thenwarmed to 0° C. for 15 min. The reaction was quenched with saturatedNH₄Cl. The mixture was partitioned between EtOAc and H₂O. The aqueouslayer was extracted with EtOAc. The combined organics were washed withbrine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the cyclosporine alkyne (364 mg, 37%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ7.86 (d, J=9.7 Hz, 1H), 7.62 (d,J=9.3 Hz, 1H), 7.56 (d, J=6.9 Hz, 1H), 7.34 (d, J=7.7 Hz, 1H), 5.73-5.68(m, 1H), 5.57-5.45 (m, 2H), 5.22-4.45 (m, 12H), 4.03-3.98 (m, 1H), 3.49(s, 3H), 3.38 (s, 3H),3.24 (s, 3H), 3.09 (s, 3H), 3.08 (s, 3H), 2.72 (s,3H), 2.70 (s, 3H), 2.50-0.64 (m, 66H); ESI MS m/z 1187[C₆₁H₁₀₇N₁₁O₁₂+H]⁺.

Example 5 Preparation of the Acetate of Cyclosporin Alkyne

To a −78° C. solution of (trimethylsilyl)diazomethane (4.5 mL, 2.0 Msolution in Et₂O, 8.9 mmol) in THF (10 mL) was added n-BuLi (3.2 mL, 2.5M solution in hexanes, 8.1 mmol) dropwise. The resulting yellowsuspension was stirred for 30 min, and then a solution of acetylcyclosporine aldehyde from Example 2 (1.00 g, 0.81 mmol) in THF (5 mL)was added dropwise. The mixture was stirred at −78° C. for 5 min. Thereaction was quenched with a mixture of acetic anhydride (1.5 mL, 4.1mmol) and pyridine (1.4 mL, 4.9 mmol) in THF (5 mL) and then warmed toroom temperature for 15 min. The reaction was quenched with saturatedNH₄Cl. The mixture was partitioned between Et₂O and H₂O. The aqueouslayer was extracted with Et₂O. The combined organics were washed withbrine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the acetate of cyclosporine alkyne (389 mg,40%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.46 (d, J=9.5 Hz, 1H),8.07 (d, J=6.9 Hz, 1H), 7.72 (d, J=9.1 Hz, 1H), 7.54 (d, J=7.7 Hz, 1H),5.69 (dd, J=10.8, 3.6 Hz, 1H), 5.55-5.40 (m, 3H), 5.30 (dd, J=11.7, 3.6Hz, 1H), 5.15 (t, J=6.1 Hz, 1H), 5.02-4.60 (m, 5H), 4.47 (t, J=6.9 Hz,1H), 3.46 (s, 3H), 3.28 (s, 3H), 3.23 (s, 3H), 3.20 (s, 3H), 3.07 (s,3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.02 (m, 5H),2.00 (s, 3H), 1.95-1.55 (m, 8H), 1.45-0.75 (m, 55H); ESI MS m/z 1229[C₆₃H₁₀₉N₁₁ _(O) ₁₃+H]⁺.

Example 6 Preparation of the Acetate of Cyclosporin Alkyne

To a solution of cyclosporine alkyne from Example 3 (0.44 g, 0.37 mmol)in methylene chloride (5 mL) was added pyridine (0.90 mL, 11.1 mmol)followed by DMAP (68 mg, 0.55 mmol) and acetic anhydride (0.70 mL, 7.4mmol), then the mixture was stirred at room temperature for 1.5 d. Thereaction mixture was diluted with ethyl ether (100 mL), washed with asaturated solution of sodium bicarbonate (30 mL) and brine (30 mL). Theorganic layer was dried over anhydrous sodium sulfate and concentratedunder vacuum. The crude material was purified by semi-preparative HPLCto afford the acetate of cyclosporine alkyne (0.23 g, 51%) as a whitesolid: ¹H NMR (300 MHz, CDCl₃) δ 8.46 (d, J=9.5 Hz, 1H), 8.07 (d, J=6.9Hz, 1H), 7.72 (d, J=9.1 Hz, 1H), 7.54 (d, J=7.7 Hz, 1H), 5.69 (dd,J=10.8, 3.6 Hz, 1H), 5.55-5.40 (m, 3H), 5.30 (dd, J=11.7, 3.6 Hz, 1H),5.15 (t, J=6.1 Hz, 1H), 5.02-4.60 (m, 5H), 4.47 (t, J=6.9 Hz, 1H), 3.46(s, 3H), 3.28 (s, 3H), 3.23 (s, 3H), 3.20 (s, 3H), 3.07 (s, 3H), 2.69(s, 3H), 2.67 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.02 (m, 5H), 2.00 (s,3H), 1.95-1.55 (m, 8H), 1.45-0.75 (m, 55H); ESI MS m/z 1229[C₆₃H₁₀₉N₁₁O₁₃+H]⁺.

Example 7 Preparation of Cyclosporin yne-ene

To a mixture of cyclosporin alkyne from Example 3 (55 mg, 0.046 mmol),copper(I) iodide (4 mg, 0.023 mmol),dichlorobis(triphenylphosphine)palladium(II) (16 mg, 0.023 mmol) intriethylamine (2 mL) was added vinyliodide (34 μL, 0.46 mmol), then themixture was stirred at room temperature for 1 h. The reaction mixturewas filtered through a micro-filter and concentrated under vacuum. Thecrude material was purified by semi-preparative HPLC to affordcyclosporin yne-ene (24 mg, 43%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 8.11 (d, J=9.3 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H), 7.56 (d, J=8.4Hz, 1H), 7.30 (d, J=7.8 Hz, 1H), 5.80-5.65 (m, 3H), 5.56 (d, J=2.4 Hz,1H), 5.52-5.36 (m, 4H), 5.30 (dd, J=11.4, 3.6 Hz, 1H), 5.20-4.95 (m,6H), 4.84 (t, J=7.2 Hz, 2H), 4.72-4.60 (m, 2H), 4.53 (t, J=7.2 Hz, 1H),3.88 (t, J=6.3 Hz, 1H), 3.50 (s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.13(s, 3H), 3.10 (s, 3H), 2.71 (s, 3H) 2.70 (s, 3H), 2.45-2.35 (m, 2H),2.20-1.80 (m, 8H), 1.75-1.55 (m, 5H), 1.45-0.75 (m, 48H); ESI MS m/z1213 [C₆₃H₁₀₉N₁₁O₁₂+H]⁺; HPLC 98.6% (AUC), t_(R)=19.32 min.

Example 8 Preparation of trans-Cyclosporin yne-ene

To a mixture of cyclosporin alkyne from Example 3 (65 mg, 0.055 mmol),copper(I) iodide (5 mg, 0.028 mmol),dichlorobis(triphenylphosphine)palladium(II) (20 mg, 0.028 mmol) intriethylamine (2 mL) was added trans-1,2-dichloroethylene (85 μL, 1.1mmol), then the mixture was stirred at room temperature for 4 h. Thereaction mixture was filtered through a micro-filter and concentratedunder vacuum. The crude material was purified by semi-preparative HPLCto afford trans-cyclosporin yne-ene (13 mg, 19%) as a white solid: ¹HNMR (300 MHz, CDCl₃) δ 8.08 (d, J=9.6 Hz, 1H), 7.71 (d, J=7.2 Hz, 1H),7.57 (d, J=8.1 Hz, 1H), 7.27 (d, J=7.8 Hz, 1H), 6.43 (d, J=13.6 Hz, 1H),5.90 (d, J=13.6 Hz, 1H), 5.70 (dd, J=10.8, 3.6 Hz, 1H), 5.45 (d, J=6.3Hz, 1H), 5.30 (dd, J=11.7, 3.6 Hz, 1H), 5.17-4.92 (m, 5H), 4.83 (t,J=6.9 Hz, 1H), 4.75-4.62 (m, 2H), 4.54 (t, J=7.2 Hz, 1H), 3.85 (t, J=6.3Hz, 1H), 3.49 (s, 3H), 3.39 (s, 3H), 3.27 (s, 3H), 3.12 (s, 3H), 3.11(s, 3H), 2.71 (s, 3H), 2.70 (s, 3H), 2.45-2.35 (m, 2H),2.20-1.80 (m,8H), 1.75-1.55 (m, 5H), 1.45-0.75 (m, 53H); ESI MS m/z 1247[C₆₃H₁₀₈ClN₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=19.92 min.

Example 9 Preparation of the Acetate of cis-Cyclosporin Yne-ene

To a mixture of the acetate of cyclosporine alkyne from Example 6 (166mg, 0.14 mmol), copper(I) iodide (13 mg, 0.068 mmol),dichlorobis(triphenylphosphine)palladium(II) (48 mg, 0.068 mmol) intriethylamine (4 mL) was added cis-1,2-dichloroethylene (0.20 mL, 2.7mmol), then the mixture was stirred at room temperature for 12 h.Cis-1,2-dichloroethylene (0.10 mL, 1.3 mmol) was refilled, and themixture was stirred for 5 h. The reaction mixture was filtered through amicro-filter and concentrated under vacuum. The crude material waspurified by semi-preparative HPLC to afford the acetate ofcis-cyclosporin yne-ene (22 mg, 13%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 8.45 (d, J=9.6 Hz, 1H), 8.08 (d, J=7.2 Hz, 1H), 7.79 (d, J=8.1Hz, 1H), 7.59 (d, J=7.8 Hz, 1H), 6.28 (d, J=7.4 Hz, 1H), 5.82 (d, J=7.4Hz, 1H), 5.70 (dd, J=10.8, 3.6 Hz, 1H), 5.62-5.10 (m, 5H), 5.03-4.72 (m,4H), 4.64 (d, J=13.8 Hz, 1H), 4.48 (t, J=7.0 Hz, 1H), 3.44 (s, 3H), 3.29(s, 3H), 3.24 (s, 3H), 3.19 (s, 3H), 3.08 (s, 3H), 2.69 (s, 3H), 2.68(s, 3H), 2.45-2.35 (m, 1H), 2.30-2.05 (m, 7H), 1.99 (s, 3H), 1.95-1.60(m, 5H), 1.45-0.75 (m, 55H); ESI MS m/z 1289 [C₆₅H₁₁₀ClN₁₁O₁₃+H]⁺.

Example 10 Preparation of cis-Cyclosporin yne-ene

To a solution of acetate of cis-cyclosporin yne-ene from Example 9 (22mg, 0.017 mmol) in MeOH (3 mL) was added potassium carbonate (47 mg,0.34 mmol), then the mixture was stirred at room temperature for 8 h.The reaction mixture was diluted with ethyl acetate (30 mL), then washedwith water (10 mL). The aqueous layer was separated and extracted withethyl acetate (30 mL). The combined organics were dried over anhydroussodium sulfate and concentrated under vacuum. The crude material waspurified by semi-preparative HPLC to afford cis-cyclosporin yne-ene (16mg, 76%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.13 (d, J=9.6 Hz,1H), 7.76 (d, J=7.2 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.33 (d, J=7.8 Hz,1H), 6.30 (d, J=7.3 Hz, 1H), 5.83 (d, J=7.3 Hz, 1H), 5.71 (dd, J=11.1,4.0 Hz, 1H), 5.44 (d, J=6.7 Hz, 1H), 5.31 (dd, J=11.5, 3.6 Hz, 1H),5.17-4.95 (m, 5H), 4.84 (t, J=7.2 Hz, 1H), 4.75-4.62 (m, 2H), 4.54 (t,J=7.2 Hz, 1H), 3.92 (t, J=6.5 Hz, 1H), 3.49 (s, 3H), 3.39 (s, 3H), 3.27(s, 3H), 3.12 (s, 3H), 3.10 (s, 3H), 2.72 (s, 3H), 2.71 (s, 3H),2.45-2.25 (m, 2H), 2.20-1.90 (m, 6H), 1.80-1.55 (m, 5), 1.45-0.75 (m,55H); ESI MS m/z 1247 [C₆₃H₁₀₈ClN₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=19.21min.

Example 11 Preparation of the Acetate of trans-Cyclosporin yne-ene

To a mixture of the acetate of cyclosporin alkyne from Example 6 (74 mg,0.06 mmol), copper(I) iodide (6 mg, 0.03 mmol),dichlorobis(triphenylphosphine)palladium(II) (21 mg, 0.03 mmol) intriethylamine (2 mL) was added (2-bromovinyl)trimethylsilane (0.18 mL,1.2 mmol), then the mixture was stirred at room temperature for 12 h.The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford the acetate of trans-cyclosporin yne-ene(16 mg, 20%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.48 (d, J=9.6Hz, 1H), 8.07 (d, J=7.2 Hz, 1H), 7.66 (d, J=8.1 Hz, 1H), 7.54 (d, J=7.8Hz, 1H), 6.24 (d, J=19.2 Hz, 1H), 5.88 (d, J=19.2 Hz, 1H), 5.70 (dd,J=10.8, 3.6 Hz, 1H), 5.55-5.10 (m, 6H), 5.03-4.92 (m, 2H), 4.86 (t,J=7.2 Hz, 1H), 4.76 (t, J=9.5 Hz, 1H), 4.64 (d, J=13.9 Hz, 1H), 4.46 (t,J=7.2 Hz, 1H), 3.44 (s, 3H), 3.31 (s, 3H), 3.25 (s, 3H), 3.20 (s, 3H),3.07 (s, 3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.05(m, 7H), 1.98 (s, 3H), 1.75-1.55 (m, 3H), 1.45-0.75 (m, 56H), 0.07 (s,9H); ESI MS m/z 1327 [C₆₈H₁₁₉N_(O) ₁₃Si+H]⁺.

Example 12 Preparation of trans-Cyclosporin yne-ene

To a solution of the acetate of trans-cyclosporin yne-ene from Example11 (16 mg, 0.012 mmol) in MeOH (2 mL) was added potassium carbonate (41mg, 0.30 mmol), then the mixture was stirred at room temperature for 8h. The reaction mixture was diluted with ethyl acetate (30 mL), thenwashed with water (10 mL). The aqueous layer was separated and extractedwith ethyl acetate (30 mL). The combined organics were dried overanhydrous sodium sulfate and concentrated under vacuum. The crudematerial was purified by semi-preparative HPLC to affordtrans-cyclosporin yne-ene (6 mg, 40%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 8.12 (d, J=9.8 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 7.58 (d, J=8.2Hz, 1H), 7.34 (d, J=7.9 Hz, 1H), 6.30 (d, J=19.2 Hz, 1H), 5.89 (d,J=19.2 Hz, 1H), 5.71 (dd, J=11.3, 4.1 Hz, 1H), 5.39 (d, J=7.1 Hz, 1H),5.30 (dd, J=11.4, 3.4 Hz, 1H), 5.20-4.95 (m, 5H), 4.83 (t, J=7.2 Hz,1H), 4.75-4.65 (m, 2H), 4.53 (t, J=7.2 Hz, 1H), 3.92 (t, J=6.4 Hz, 1H),3.49 (s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.13 (s, 3H), 3.10 (s, 3H),2.72 (s, 3H), 2.71 (s, 3H), 2.60-2.35 (m, 2H), 2.20-1.55 (m, 12H),1.45-0.75 (m, 54H), 0.07 (s, 9H); ESI MS m/z 1285 [C₆₆H₁₁₇N₁₁O₁₂Si+H]⁺;HPLC 98.5% (AUC), t_(R)=22.71 min.

Example 13 Preparation of trans-Cyclosporin yne-ene

To a mixture of cyclosporine alkyne from Example 3 (40 mg, 0.03 mmol) intriethylamine (2 mL) and tetrahydrofuran (1 mL) was addeddichlorobis(triphenylphosphine)palladium(II) (15 mg, 0.02 mmol),copper(I) iodide (4 mg, 0.02 mmol) and trans-1-bromopropene (50 μL, 0.6mmol), then the mixture was stirred at room temperature for 3 h. Thereaction mixture was filtered through a micro-filter and concentratedunder vacuum. Purification twice by semi-preparative HPLC gavetrans-cyclosporin yne-ene (5.5 mg, 15%) as a yellow solid: ¹H NMR (300MHz, CDCl₃) δ 8.10 (d, J=5.6 Hz, 1H), 7.75-7.67 (m, 3H), 7.49-7.38 (m,4H), 6.05-5.99 (m, 1H), 5.70 (dd, J=10.9, 4.2 Hz, 1H), 5.42 (dd, J=15.8,1.8 Hz, 1H), 5.36 (d, J=7.0 Hz, 1H), 5.28 (dd, J=11.4, 3.5 Hz, 1H),5.19-5.17 (m, 1H), 5.09 (t, J=6.7 Hz, 1H), 5.064.98 (m, 3H), 4.84 (t,J=7.2 Hz, 1H), 4.73-4.68 (m, 3H), 4.49 (t, J=7.3 Hz, 1H), 3.90 (t, J=6.6Hz, 1H), 3.49 (s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.13 (s, 3H), 3.09(s, 3H), 2.71 (s, 3H), 2.70 (s, 3H), 2.55-1.80 (m, 12H), 1.75-1.56 (m,13H), 1.50-1.19 (m, 41H); ESI MS m/z 1227 [C₆₄H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99%(AUC), t_(R)=19.76 min.

Example 14 Preparation of cis-Cyclosporin yne-ene

A mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol),cis-1-bromopropene (300 μL, 3.5 mmol) and copper(I) iodide (14 mg, 0.07mmol) in triethylamine (3 mL) was stirred until a clear solution formed.Dichlorobis(triphenylphosphine)palladium(II) (51 mg, 0.07 mmol) wasadded, and then the mixture was stirred at room temperature overnight.The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The residue was purified by columnchromatography (silica gel, 7:3 hexanes/ethyl acetate to ethyl acetate)to give a brown solid. The solid was further purified twice bysemi-preparative HPLC to afford cis-cyclosporin yne-ene (29 mg, 35%) asa white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.15 (d, J=9.5 Hz, 1H), 7.77(d, J=7.3 Hz, 1H), 7.51 (d, J=8.5 Hz, 1H), 7.27 (d, J=6.4 Hz, 1H),5.94-5.83 (m, 1H), 5.70 (dd, J=11.0, 4.2 Hz, 1H), 5.45-5.39 (m, 2H),5.29 (dd, J=11.5, 3.8 Hz, 1H), 5.20-5.16 (m, 1H), 5.10-4.97 (m, 4H),4.88-4.79 (m, 1H), 4.7144.67 (m, 3H), 4.54-4.47 (m 1H), 3.90 (t, J=6.5Hz, 1H), 3.50 (s, 3H), 3.39 (s, 3H), 3.27 (s, 3H), 3.13 (s, 3H), 3.10(s, 3H), 2.71 (s, 3H), 2.70 (s, 3H), 2.48-0.80 (m, 70H); ESI MS m/z 1227[C₆₄H₁₁₁N₁₁O₁₂+H]⁺, HPLC >99% (AUC), t_(R)=19.85 min.

Example 15 Preparation of Cyclosporin yne-ene

To a mixture of cyclosporin alkyne from Example 3 (40 mg, 0.03 mmol) intriethylamine (2 mL) and tetrahydrofuran (1 mL) was addeddichlorobis(triphenylphosphine)palladium(II) (15 mg, 0.02 mmol),copper(I) iodide (4 mg, 0.02 mmol) and 2-bromopropene (50 μL, 0.6 mmol),then the mixture was stirred at room temperature for 5 h. The reactionmixture was filtered through a micro-filter and concentrated undervacuum. Purification twice by semi-preparative HPLC gave cyclosporinyne-ene (4.5 mg, 12%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.12(d, J=9.0 Hz, 1H), 7.74 (d, J=7.3 Hz, 1H), 7.49 (d, J=8.3 Hz, 1H), 7.22(d, J=7.8 Hz, 1H), 5.71 (dd, J=10.9, 4.3 Hz, 1H), 5.41 (d, J=6.8 Hz,1H), 5.35 (dt, J=9.1, 5.8 Hz, 1H), 5.27 (dd, J=11.5, 3.7 Hz, 1H),5.20-5.18 (m, 3H), 5.14 (s, 1H), 5.08 (t, J=6.9 Hz, 1H), 5.05-4.98 (m,2H), 4.84 (app quintet, J=6.9 Hz, 1H), 4.73-4.67 (m, 2H), 4.52 (appquintet, J=7.4 Hz, 1H), 3.88 (t, J=6.5 Hz, 1H ), 3.50 (s, 3H), 3.38 (s,3H), 3.28 (s, 3H), 3.21-3.18 (m, 1H), 3.13 (s, 3H), 3.10 (s, 3H), 2.71(s, 3H), 2.70 (s, 3H), 2.60 (dd, J=17.1, 3.9 Hz, 1H), 2.46-2.35 (m, 1H),2.63 (t, J=7.7 Hz, 1H), 2.18-2.07 (m, 6H), 2.05-1.96 (m, 3H), 1.90-1.82(m, 5H), 1.81-1.59 (m, 5H), 1.52-1.38 (m, 4H), 1.36-1.23 (m, 13H),1.01-0.84 (m, 30H); ESI MS m/z 1227 [C₆₄H₁₁₁N₁₁O₁₂+H]⁺; HPLC 98.8%(AUC), t_(R=)19.82 min.

Example 16 Preparation of Cyclosporin yne-ene

To a mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol) andcopper(I) iodide (13 mg, 0.07 mmol) in triethylamine (3 mL) was addedbromostyrene (a mixture of cis and trans isomers, 180 μL, 1.4 mmol) thenthe mixture was stirred until a clear solution formed.Dichlorobis(triphenylphosphine)palladium(II) (50 mg, 0.07 mmol) wasadded, and then the mixture was stirred at room temperature overnight.The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The residue was purified by columnchromatography (silica gel, 7:3 hexanes/ethyl acetate to ethyl acetate)to give a brown solid. The solid was further purified twice bysemi-preparative HPLC to afford cyclosporin yne-ene (17 mg, 19%) as awhite solid and a mixture of isomers (cis/trans˜1:4 by ¹H NMR): ¹H NMR(500 MHz, CDCl₃) δ 8.10 (d, J=9.7 Hz, 1H), 7.81 (d, J=7.4 Hz, 1H), 7.71(d, J=7.4 Hz, 1H), 7.47 (d, J=8.5 Hz, 1H), 7.38-7.29 (m, 12H), 6.94-6.83(m, 4H), 5.70 (dd, J=11.0, 4.3 Hz, 1H), 5.44 (d, J=6.9 Hz, 2H),5.19-5.15 (m, 2H), 5.08 (t, J=7.3 Hz, 1H), 5.06-5.00 (m, 6H), 4.83 (t,J=7.7 Hz, 1H), 4.73-4.69 (m, 4H), 4.55-4.40 (m, 3H), 3.92 (t, J=6.6 Hz,1H), 3.63 (s, 3H), 3.52 (s, 3H), 3.40 (s, 3H), 3.29 (s, 3H), 3.12 (s,3H), 3.11 (s, 3H), 2.50-1.87 (m, 10H), 1.84-1.57 (m, 3H), 1.55-1.19 (m,10H), 1.08-0.70 (m, 31H); ESI MS m/z 1289 [C₆₉H₁₁₃N_(O) ₁₂+H]⁺; HPLC98.2% (AUC), t_(R)=20.66 min.

Example 17 Preparation of Phenyl Cyclosporin Alkyne

A mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol) intriethylamine (3 mL) was degassed with N₂ for 5 min.Dichlorobis(triphenylphosphine)palladium(II) (28 mg, 0.04 mmol),copper(I) iodide (8 mg, 0.04 mmol) and iodobenzene (80 μL, 0.70 mmol)were added, then the mixture was stirred at room temperature for 1.5 h.The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. Purification by semi-preparative HPLC gavecyclosporine phenyl alkyne (7 mg, 8%) as a brown solid: ¹H NMR (500 MHz,CDCl₃) δ 8.18 (d, J=9.8 Hz, 1H), 7.78 (d, J=7.4 Hz, 1H), 7.71-7.68 (m,1H), 7.51 (d, J=8.3 Hz, 1H), 7.40-7.37 (m, 4H), 7.20 (d, J=7.9 Hz, 1H),5.69 (dd, J=10.6, 3.7 Hz, 1H), 5.45 (d, J=6.9 Hz, 1H), 5.32-5.27 (m,1H), 5.20-5.16 (m, 1H), 5.09-4.97 (m, 4H), 4.83 (t, J=7.1 Hz, 1H),4.74-4.67 (m, 2H), 4.52 (t, J=7.3 Hz, 1H), 3.92 (t, J=6.5 Hz, 1H), 3.53(s, 3H), 3.40 (s, 3H), 3.30 (s, 3H), 3.22-3.17 (m, 2H), 3.13 (s, 3H),3.10 (s, 3H), 2.70 (s, 3H), 2.69 (s, 3H), 2.53-2.35 (m, 2H), 2.29-1.91(m, 10H), 1.83-1.83 (m, 11H), 1.50-1.18 (m, 11H), 1.10-0.76 (m, 32H);ESI MS m/z 1263 [C₆₇H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=20.01 min.

Example 18 Preparation of 4-Methoxyphenyl Cyclosporin Alkyne

A mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol),1-iodo-4-methoxybenzene (150 μL, 1.4 mmol) and copper(I) iodide (13 mg,0.07 mmol) in triethylamine (3 mL) was stirred until a clear solutionformed. Dichlorobis(triphenylphosphine)palladium(II) (50 mg, 0.07 mmol)was added, and then the mixture was stirred at room temperatureovernight. The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The residue was purified by columnchromatography (silica gel, 7:3 hexanes/ethyl acetate to ethyl acetate)to give a brown solid. The solid was further purified twice bysemi-preparative HPLC to afford 4-methoxyphenyl cyclosporin alkyne (23mg, 26%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.17 (d, J=9.7 Hz,1H), 7.77 (d, J=7.4 Hz, 1H), 7.50 (d, J=8.5 Hz, 1H), 7.31 (d, J=8.8 Hz,2H), 7.21 (d, J=7.9 Hz, 1H), 6.80 (d, J=8.8 Hz, 2H), 5.68 (dd, J=11.0,4.3 Hz, 1H), 5.42 (d, J=7.1 Hz, 1H), 5.29 (dd, J=11.6, 3.9 Hz, 1H),5.20-5.18 (m, 2H), 5.09-5.05 (m, 2H), 5.01 (dd, J=16.2, 8.1 Hz, 1H),4.85 (app quintet, J=6.9 Hz, 1H), 4.73-4.69 (m, 2H), 4.51 (app quintet,J=7.2 Hz, 1H), 3.92 (d, J=6.4 Hz, 1H), 3.80 (s, 3H), 3.52 (s, 3H), 3.40(s, 3H), 3.29 (s, 3H), 3.20-3.14 (m, 6H), 3.13 (s, 3H), 3.10 (s, 3H),2.69 (s, 3H), 2.67 (s, 3H), 2.44-1.95 (m, 12H), 1.76-1.61 (m, 3H),1.45-1.24 (m, 16H), 1.07 (t, J=8.0 Hz, 4H), 0.97-0.79 (m, 27H); ESI MSm/z 1293 [C₆₈H₁₁₃N₁₁O₁₃+H]⁺; HPLC >99% (AUC), t_(R)=19.59 min.

Example 19 Preparation of 4-Fluorophenyl Cyclosporin Alkyne

A mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol),4-fluoro-1-iodobenzene (160 μL, 1.4 mmol) and copper(I) iodide (14 mg,0.07 mmol) in triethylamine (3 mL) was stirred until a clear solutionformed. Dichlorobis(triphenylphosphine)palladium(II) (50 mg, 0.07 mmol)was added, and then the mixture was stirred at room temperatureovernight. The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The residue was purified by columnchromatography (silica gel, 7:3 hexanes/ethyl acetate to ethyl acetate)to give a brown solid. The solid was further purified twice bysemi-preparative HPLC to afford 4-fluorophenyl cyclosporin alkyne (8.9mg, 10%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 8.14 (d, J=9.8 Hz,1H), 7.76 (d, J=7.4 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 7.35 (ddd, J=8.7,5.4, 2.0 Hz, 2H), 7.19 (d, J=7.9 Hz, 1H), 6.97 (dd, J=8.7, 8.7 Hz, 2H),5.68 (dd, J=11.1, 4.1 Hz, 1H), 5.45 (d, J =6.7 Hz, 1H), 5.27 (dd,J=11.5, 3.7 Hz, 1H), 5.18-5.14 (m, 1H), 5.09-4.94 (m, 4H), 4.88-4.79 (m,1H), 4.74-4.66 (m, 2H), 4.56-4.47 (m, 1H), 3.91 (t, J=6.4 Hz, 1H), 3.52(s, 3H), 3.40 (s, 3H), 3.29 (s, 3H), 3.13 (s, 3H), 3.10 (s, 3H), 2.71(s, 3H), 2.69 (s, 3H), 2.45-0.76 (m, 68H); ESI MS m/z 1280[C₆₇H₁₁₀FN₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=20.18 min.

Example 20 Preparation of Thiophen-2-yl Cyclosporin Alkyne

A mixture of cyclosporin alkyne from Example 3 (80 mg, 0.07 mmol),2-iodothiophene (328 mg, 1.4 mmol) and copper(I) iodide (13 mg, 0.07mmol) in triethylamine (3 mL) was stirred until a clear solution formed.Dichlorobis(triphenylphosphine)palladium(II) (50 mg, 0.07 mmol) wasadded, and then the mixture was stirred at room temperature overnight.The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The residue was purified by columnchromatography (silica gel, 7:3 hexanes/ethyl acetate to ethyl acetate)to give a light brown solid. The solid was further purified twice bysemi-preparative HPLC to afford thiophen-2-yl cyclosporin alkyne (10.7mg, 12%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.07 (d, J=9.6 Hz,1H), 7.74 (d, J=7.4 Hz, 1H), 7.54 (d, J=8.3 Hz, 1H), 7.25 (d, J=5.6 Hz,1H), 7.15 (dd, J=5.2, 1.0 Hz, 1H), 7.11 (dd, J=3.5, 0.9 Hz, 1H), 6.92(dd, J=5.1, 3.6 Hz, 1H), 5.69 (dd, J =11.0, 4.3 Hz, 1H), 5.47 (d, J=6.4Hz, 1H), 5.29 (dd, J=11.4, 3.6 Hz, 1H), 5.18-5.16 (m, 1H), 5.07 (t,J=6.9 Hz, 1H), 5.05-5.00 (m, 2H), 4.86-4.81 (m, 1H), 4.74-4.67 (m, 2H),4.57-4.51 (m, 1H), 3.90 (t, J=6.4 Hz, 1H), 3.52 (s, 3H), 3.39 (s, 3H),3.28 (s, 3H), 3.25-3.15 (m, 3H), 3.12 (s, 3H), 3.10 (s, 3H), 3.07-2.90(m, 4H), 2.82-2.75 (m, 1H), 2.72 (s, 3H), 2.70 (s, 3H), 2.44-1.90 (m,8H), 1.79-1.58 (m, 6H), 1.48-1.18 (m, 11H), 1.05-0.80 (m, 36H); ESI MSm/z 1269 [C₆₅H₁₀₉N₁₁O₁₂S+H]⁺; HPLC 98.8% (AUC), t_(R)=19.76 min.

Example 21 Preparation of the Acetate of Cyclosporin Diyne

To a solution of the acetate of cyclosporin alkyne from Example 6 (90mg, 0.073 mmol) in pyrrolidine (1 mL) were added copper(I) iodide (7 mg,0.037 mmol) and dichlorobis(triphenylphosphine)palladium(II) (26 mg,0.037 mmol), then the mixture was stirred for 5 min at room temperature.1-Butynyl iodide (145 μL, 1.46 mmol) was added dropwise, and then themixture was stirred overnight at room temperature. The reaction mixturewas diluted with ethyl acetate (40 mL) and washed with a saturatedsolution of ammonium chloride (20 mL). The aqueous layer was extractedwith ethyl acetate (2×20 mL). The combined organics were dried overanhydrous sodium sulfate and concentrated under vacuum. The crudematerial was purified by semi-preparative HPLC to afford the desiredacetate of cyclosporin diyne (15 mg, 16%) as a brown solid: ¹H NMR (300MHz, CDCl₃) δ 8.48 (d, J=9.6 Hz, 1H), 8.05 (d, J=6.9 Hz, 1H), 7.60 (d,J=9.0 Hz, 1H), 7.55 (d, J=7.8 Hz, 1H), 5.70 (dd, J=10.8, 3.9 Hz, 1H),5.58-5.35 (m, 2H), 5.30 (dd, J=12.0, 3.3 Hz, 2H), 5.15 (t, J=6.9 Hz,1H), 5.05-4.80 (m, 3H), 4.73 (t, J=9.6 Hz, 1H), 4.64 (d, J=13.8 Hz, 1H),4.44 (t, J=6.9 Hz, 1H), 3.43 (s, 3H), 3.32 (s, 3H), 3.27 (s, 3H), 3.20(s, 3H), 3.07 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H), 2.50-2.35 (m, 1H),2.30-1.80 (m, 10H), 2.04 (s, 3H), 1.75-1.55 (m, 3H), 1.45-0.75 (m, 59H);ESI MS m/z 1281 [C₆₇H₁₁₃N₁₁O₁₃+H]⁺.

Example 22 Preparation of Cyclosporin Diyne

To a solution of the acetate of cyclosporin diyne from Example 21 (18mg, 0.014 mmol) in MeOH (2 mL) was added potassium carbonate (39 mg,0.28 mmol), then the mixture was stirred overnight at room temperature.The reaction mixture was quenched with a saturated solution of ammoniumchloride, and then extracted with ethyl acetate (3×30 mL). The combinedorganics were dried over anhydrous sodium sulfate and concentrated undervacuum. The crude material was purified by semi-preparative HPLC toafford cyclosporin diyne (9 mg, 53%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 7.98 (d, J=9.3 Hz, 1H), 7.73 (d, J=7.2 Hz, 1H), 7.39 (d, J=8.4Hz, 1H), 7.23 (d, J=8.1 Hz, 1H), 5.71 (dd, J=10.8, 3.9 Hz, 1H), 5.41 (d,J=6.6 Hz, 1H), 5.28 (dd, J=11.7, 3.9 Hz, 1H), 5.20-4.95 (m, 5H), 4.83(t, J=7.2 Hz, 1H), 4.78-4.63 (m, 2H), 4.52 (t, J=7.2 Hz, 1H), 3.90 (t,J=6.3 Hz, 1H), 3.50 (s, 3H), 3.37 (s, 3H), 3.28 (s, 3H), 3.12 (s, 3H),3.09 (s, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.55-2.05 (m, 7H), 1.90-0.80(m, 66H); ESI MS m/z 1238 [C₆₅H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC),t_(R)=20.06 min.

Example 23 Preparation of the Acetate of Cyclosporin Alkynyl Bromide

To a solution of the acetate of cyclosporin alkyne from Example 6 (0.22g, 0.18 mmol) in acetone (5 mL) was added N-bromosuccinimide (64 mg,0.36 mmol) followed by silver nitrate (6 mg, 0.036 mmol). The reactionflask was wrapped with aluminum foil. The reaction mixture was stirredat room temperature for 1 h, then poured into ice-water (20 mL) andextracted with ethyl ether (3×40 mL). The combined organics were washedwith brine (30 mL), dried over anhydrous sodium sulfate and concentratedunder vacuum. The crude material was purified by semi-preparative HPLCto afford the acetate of cyclosporin alkynyl bromide (0.23 g, 98%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ 8.48 (d, J=9.6 Hz, 1H), 8.07 (d,J=6.9 Hz, 1H), 7.80 (d, J=8.9 Hz, 1H), 7.63 (d, J=7.7 Hz, 1H), 5.70 (dd,J=10.8, 3.6 Hz, 1H), 5.60-5.15 (m, 5H), 5.02-4.80 (m, 4H), 4.76 (d,J=9.3 Hz, 1H), 4.65 (d, J=13.9 Hz, 1H), 4.48 (t, J=7.0 Hz, 1H), 3.43 (s,3H), 3.30 (s, 3H), 3.25 (s, 3H), 3.19 (s, 3H), 3.10 (s, 3H), 2.69 (s,3H), 2.68 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.05 (m, 7H), 2.02 (s, 3H),1.75-1.55 (m, 4H), 1.45-0.75 (m, 55H); ESI MS m/z 1307[C₆₃H₁₀₈BrN₁₁O₁₃+H]⁺.

Example 24 Preparation of the Acetate ofCyclosporin(trimethylsilyl)diyne

To a solution of the acetate of cyclosporin alkynyl bromide from Example23 (20 mg, 0.015 mmol) in pyrrolidine (1 mL) was added(trimethylsilyl)acetylene (42 μL, 0.30 mmol) followed by copper(I)iodide (3 mg, 0.015 mmol) anddichlorobis(triphenylphosphine)palladium(II) (6 mg, 0.008 mmol), thenthe mixture was stirred at room temperature for 1 h. The reaction wasquenched with a saturated solution of ammonium chloride, and thenextracted with ethyl acetate (3×30 mL). The combined organics were driedover anhydrous sodium sulfate and concentrated under vacuum. The crudematerial was purified by semi-preparative HPLC to afford the acetate ofcyclosporin (trimethylsilyl)diyne (6 mg, 30%) as a pale-brown solid: ¹HNMR (300 MHz, CDCl₃) δ 8.48 (d, J=9.6 Hz, 1H), 8.05 (d, J=6.7 Hz, 1H),7.65 (d, J=8.8 Hz, 1H), 7.57 (d, J=7.9 Hz, 1H), 5.70 (dd, J=11.2, 3.8Hz, 1H), 5.55-5.35 (m, 2H), 5.29 (td, J=11.9, 3.7 Hz, 2H), 5.16 (d,J=6.1 Hz, 1H), 5.03-4.80 (m, 3H), 4.72 (t, J=9.4 Hz, 11H), 4.63 (d,J=13.9 Hz, 1H), 4.46 (t, J=7.0 Hz, 1H), 3.42 (s, 3H), 3.32 (s, 3H), 3.27(s, 3H), 3.20 (s, 3H), 3.08 (s, 3H), 2.68 (s, 3H), 2.67 (s, 3H),2.45-2.05 (m, 7H), 2.04 (s, 3H), 1.75-1.55 (m, 3H), 1.45-0.75 (m, 56H),0.15 (s, 9H); ESI MS m/z 1325 [C₆₈H₁₁₇N₁₁O₁₃Si+H]⁺.

Example 25 Preparation of Cyclosporin Diyne

To a solution of the acetate of cyclosporin (trimethylsilyl)diyne fromExample 24 (9 mg, 0.007 mmol) in MeOH (2 mL) was added potassiumcarbonate (19 mg, 0.14 mmol), then the mixture was stirred overnight atroom temperature. The reaction mixture was quenched with a saturatedsolution of ammonium chloride, and then extracted with ethyl acetate(3×30 mL). The combined organics were dried over anhydrous sodiumsulfate and concentrated under vacuum. The crude material was purifiedby semi-preparative HPLC to afford cyclosporine diyne (6 mg, 71%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ 7.95 (d, J=9.7 Hz, 1H), 7.65 (d,J=7.4 Hz, 1H), 7.38 (d, J=8.3 Hz, 1H), 7.17 (d, J=7.9 Hz, 1H), 5.71 (dd,J=10.8, 3.8 Hz, 1H), 5.48 (d, J=6.2 Hz, 1H), 5.27 (dd, J=11.5, 3.9 Hz,1H), 5.15-4.95 (m, 5H), 4.83 (t, J=7.1 Hz, 1H), 4.75-4.62 (m, 2H), 4.53(t, J=7.2 Hz, 1H), 3.89 (t, J=6.2 Hz, 1H), 3.51 (s, 3H), 3.38 (s, 3H),3.28 (s, 3H), 3.11 (s, 3H), 3.10 (s, 3H), 2.70 (s, 3H), 2.69 (s, 3H),2.60-2.35 (m, 2H), 2.20-0.80 (m, 67H); ESI MS m/z 1211[C₆₃H₁₀₇N₁₁O₁₂+H]⁺; HPLC 98.5% (AUC), t_(R)=18.78 min.

Example 26 Preparation of the Acetate of Cyclosporin Diyne

To an ice-cooled solution of 1-propynylmagnesium bromide (0.54 mL, 0.5 Min THF, 0.27 mmol) in THF (1 mL) was added a solution of zinc chloride(0.27 mL, 1 M in ethyl ether, 0.27 mmol). The reaction was stirred at 0°C. for 10 min, and then allowed to warm to room temperature. A solutionof the acetate of cyclosporin alkynyl bromide from Example 23 (35 mg,0.027 mmol) in THF (1 mL) was added into the reaction mixture followedby dichlorobis(triphenylphosphine)palladium(II) (10 mg, 0.014 mmol). Theresulting reaction mixture was stirred at room temperature for 1.5 h,and then quenched with a saturated solution of ammonium chloride (10mL). The aqueous layer was extracted with ethyl acetate (3×20 mL). Thecombined organics were dried over anhydrous sodium sulfate andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford the acetate of cyclosporin diyne (14 mg,41%) as a pale-brown solid: ¹H NMR (500 MHz, CDCl₃) δ 8.42 (d, J=9.5 Hz,1H), 8.07 (d, J=7.0 Hz, 1H), 7.82 (d, J=9.0 Hz, 1H), 7.64 (d, J=7.5 Hz,1H), 5.71 (dd, J=11.0, 4.5 Hz, 1H), 5.58-5.37 (m, 3H), 5.27 (dd, J=12.0,4.0 Hz, 1H), 5.17 (t, J=6.0 Hz, 1H), 5.03-4.93 (m, 3H), 4.87 (t, J=7.0Hz, 1H), 4.81 (t, J=9.5 Hz, 1H), 4.65 (d, J=13.5 Hz, 1H), 4.49 (t, J=7.0Hz, 1H), 3.43 (s, 3H), 3.29 (s, 3H), 3.23 (s, 3.19 (s, 3H), 3.09 (s,3H), 2.70 (s, 3H), 2.68 (s, 3H), 2.45-2.35 (m, 1H), 2.28-2.04 (m, 7H),2.03 (s, 3H), 2.02-1.92 (m, 2H), 1.88 (s, 3H), 1.75-1.62 (m, 4H),1.45-0.75 (m, 53H); ESI MS m/z 1267 [C₆₆H₁₁₁N₁₁O₁₃+H]⁺.

Example 27 Preparation of Cyclosporin Diyne

To a solution of the acetate of cyclosporin diyne from Example 26 (14mg, 0.011 mmol) in MeOH (2 mL) was added potassium carbonate (30 mg,0.22 mmol), then the mixture was stirred overnight at room temperature.The reaction mixture was quenched with a saturated solution of ammoniumchloride, and then extracted with ethyl acetate (3×30 mL). The combinedorganics were dried over anhydrous sodium sulfate and concentrated undervacuum. The crude material was purified by semi-preparative HPLC toafford cyclosporin diyne (8 mg, 62%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 7.93 (d, J=9.5 Hz, 1H), 7.71 (d, J=7.0 Hz, 1H), 7.51 (d, J=8.2Hz, 1H), 7.37 (d, J=7.4 Hz, 1H), 5.71 (dd, J=11.0, 2.9 Hz, 1H), 5.41 (d,J=6.5 Hz, 1H), 5.38-5.27 (m, 1H), 5.15-4.95 (m, 4H), 4.84 (t, J=7.0 Hz,1H), 4.75-4.65 (m, 2H), 4.54 (t, J=6.9 Hz, 1H), 3.94 (t, J=6.5 Hz, 1H),3.48 (s, 3H), 3.37 (s, 3H), 3.25 (s, 3H), 3.12 (s, 3H), 3.10 (s, 3H),2.72 (s, 3H), 2.71 (s, 3H), 2.55-2.05 (m, 7H), 1.88 (s, 3H), 1.75-0.75(m, 62H); ESI MS m/z 1225 [C₆₄H₁₀₉N₁₁O₁₂+H]⁺; HPLC >99% (AUC),t_(R)=19.38 min.

Example 28 Preparation of the Acetate of Cyclosporin Cyclopropyl Diyne

To an ice-cooled solution of cyclopropyl(trimethylsilyl)acetylene (37mg, 0.27 mmol) in triethylamine (1 mL) was added tetrabutylammoniumfluoride (0.32 mL, 1 M in THF, 0.32 mmol), then the mixture was stirredfor 10 min. The reaction mixture was allowed to warm to roomtemperature, then a solution of the acetate of cyclosporin alkynylbromide from Example 23 (35 mg, 0.027 mmol) in triethylamine (1 mL) wasadded into the mixture followed by copper(I) iodide (3 mg, 0.014 mmol)and dichlorobis(triphenylphosphine)palladium(II) (10 mg, 0.014 mmol).The resulting reaction mixture was stirred at room temperature for 5 h.The reaction mixture was diluted with ethyl acetate (30 mL) and washedwith a saturated solution of ammonium chloride (10 mL). The aqueouslayer was extracted with ethyl acetate (2×20 mL). The combined organicswere dried over anhydrous sodium sulfate and concentrated under vacuum.The crude material was purified by semi-preparative HPLC to afford theacetate of cyclosporin cyclopropyl diyne (23 mg, 66%) as a pale-brownsolid: ¹H NMR (300 MHz, CDCl₃) δ 8.45 (d, J=9.4 Hz, 1H), 8.05 (d, J=6.8Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 5.70 (dd,J=10.9, 3.6 Hz, 1H), 5.58-5.23 (m, 4H), 5.16 (d, J=5.5 Hz, 1H),5.02-4.80 (m, 3H), 4.77 (t, J=9.6 Hz, 1H), 4.64 (d, J=13.9 Hz, 1H), 4.46(t, J=7.0 Hz, 1H), 3.43 (s, 3.30 (s, 3H), 3.25 (s, 3H), 3.19 (s, 3H),3.08 (s, 3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.03(m, 6H), 2.02 (s, 3H), 1.75-1.60 (m, 3H), 1.45-0.70 (m, 63H); ESI MS m/z1293 [C₆₈H₁₁₃N₁₁O₁₃+H]⁺.

Example 29 Preparation of Cyclosporin Cyclopropyl Diyne

To a solution of the acetate of cyclosporin cyclopropyl diyne fromExample 28 (20 mg, 0.015 mmol) in MeOH (2 mL) was added potassiumcarbonate (41 mg, 0.30 mmol), then the mixture was stirred overnight atroom temperature. The reaction mixture was quenched with a saturatedsolution of ammonium chloride, and then extracted with ethyl acetate(3×30 mL). The combined organics were dried over anhydrous sodiumsulfate and concentrated under vacuum. The crude material was purifiedby semi-preparative HPLC to afford cyclosporin cyclopropyl diyne (16 mg,84%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.97 (d, J=9.7 Hz, 1H),7.70 (d, J=7.3 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.25 (d, J=8.7 Hz, 1H),5.71 (dd, J=11.0, 4.0 Hz, 1H), 5.41 (d, J=6.6 Hz, 1H), 5.28 (dd, J=11.4,3.9 Hz, 1H), 5.15-4.95 (m, 4H), 4.83 (t, J=7.2 Hz, 1H), 4.75-4.65 (m,3H), 4.52 (t, J=7.2 Hz, 1H), 3.90 (t, J=6.4 Hz, 1H), 3.49 (s, 3H), 3.37(s, 3H), 3.28 (s, 3H), 3.12 (s, 3H), 3.09 (s, 3H), 2.71 (s, 3H), 2.70(s, 3H), 2.55-2.30 (m, 2H), 2.20-0.75 (m, 71H); ESI MS m/z 1251[C₆₆H₁₁₁N₁₁O₁₂ +H]⁺; HPLC >99% (AUC), t_(R)═19.98 min.

Example 30 Preparation of the Acetate of Cyclosporin Diyne

A mixture of the acetate of cyclosporine alkynyl bromide from Example 23(40 mg, 0.03 mmol), dichlorobis(triphenylphosphine)palladium(II) (14 mg,0.02 mmol), copper(I) iodide (4 mg, 0.02 mmol) and phenylacetylene (30μL, 0.30 mmol) in triethylamine (2 mL) was stirred at room temperatureovernight. The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. Purification by column chromatography (silicagel, 100% hexanes followed by 100% EtOAc) followed by semi-preparativeHPLC gave the acetate of cyclosporin diyne (11 mg, 27%) as a brownsolid: ¹H NMR (300 MHz, CDCl₃) δ 8.52 (d, J=9.7 Hz, 1H), 8.05 (d, J=6.9Hz, 1H), 7.62-7.54 (m, 2H), 7.47-7.44 (m, 2H), 7.34-7.28 (m, 3H), 5.69(dd, J=10.9, 3.9 Hz, 1H), 5.59-5.50 (m, 1H), 5.47-5.43 (m, 1H), 5.35 (t,J=3.5 Hz, 1H), 5.31 (t, J=3.4 Hz, 1H), 5.15 (t, J=6.2 Hz, 1H), 5.02-4.91(m, 4H), 4.86 (t, J=7.2 Hz, 1H), 4.75 (t, J=9.6 Hz, 1H), 4.67-4.63 (m,1H), 4.45 (t, J=7.0 Hz, 1H), 3.44 (s, 3H), 3.42 (s, 3H), 3.28 (s, 3H),3.20 (s, 3H), 3.08 (s, 3H), 2.69 (s, 3H), 2.66 (s, 3H), 2.52-2.11 (m,8H), 2.07 (s, 3H), 1.75-1.55 (m, 4H), 1.41-1.18 (m, 20H), 1.05-0.82 (m,34H); ESI MS m/z 1329 [C₇₁H₁₁₃N₁₁O₁₃+H]⁺.

Example 31 Preparation of Cyclosporin Diyne

To a solution of the acetate of cyclosporin diyne from Example 30 (11mg, 0.008 mmol) in MeOH (1 mL) was added potassium carbonate (11 mg,0.08 mmol) and then the mixture was stirred at room temperatureovernight. The mixture was diluted with EtOAc, washed with H₂O (2×),brine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the cyclosporin diyne (6.6 mg, 64%) as abrown solid: ¹ H NMR (300 MHz, CDCl₃) δ 7.98 (d, J=9.7 Hz, 1H), 7.69 (d,J=7.5 Hz, 1H), 7.50-7.47 (m, 2H), 7.41 (d, J=8.3 Hz, 1H), 7.32-7.28 (m,3H), 7.22 (d, J=7.9 Hz, 1H), 5.70 (dd, J=11.0, 4.1 Hz, 1H), 5.48 (d,J=6.4 Hz, 1H), 5.34-5.29 (m, 2H), 5.15-4.99 (m, 6H), 4.83 (t, J=7.0 Hz,1H), 4.74-4.66 (m, 2H), 4.52 (t, J=7.3 Hz, 1H), 3.93 (t, J=6.3 Hz, 1H),3.52 (s, 3H), 3.39 (s, 3H), 3.26 (s, 3H), 3.24-3.17 (m, 3H), 3.11 (s,3H), 2.71 (s, 3H), 2.70 (s, 3H), 2.31-2.06 (m, 12H), 1.67-1.62 (m, 4H),1.55-1.21 (m, 12H), 1.07-0.84 (m, 38H); ESI MS m/z 1287[C₆₉H₁₁₁N₁₁O₁₂+H]⁺; HPLC 96.6% (AUC), t_(R)=20.88 min.

Example 32 Preparation of the Acetate of Cyclosporin yne-yne-ene

To an ice-cooled solution of the acetate of cyclosporin(trimethylsilyl)diyne from Example 24 (25 mg, 0.019 mmol) intriethylamine (1 mL) was added tetrabutylammonium fluoride (95 μL, 1 Min THF, 0.095 mmol), then the mixture was stirred for 10 min. Thereaction mixture was allowed to warm to room temperature, then copper(I)iodide (4 mg, 0.019 mmol) anddichlorobis(triphenylphosphine)palladium(II) (13 mg, 0.019 mmol) wereadded into the mixture followed by vinyl iodide (30 μL, 0.38 mmol). Theresulting reaction mixture was stirred at room temperature for 1 h. Thereaction mixture was filtered through a micro-filter and concentratedunder vacuum. The crude material was purified by semi-preparative HPLCto afford the desired acetate of cyclosporin yne-yne-ene (6 mg, 25%) asa brown oil: ¹H NMR (300 MHz, CDCl₃) δ 8.43 (d, J=9.5 Hz, 1H), 8.07 (d,J=6.7 Hz, 1H), 7.82 (d, J=8.9 Hz, 1H), 7.64 (d, J=7.7 Hz, 1H), 5.80-5.65(m, 4H), 5.60-5.52 (m, 3H), 5.45-5.33 (m, 2H), 5.28(dd, J=11.9, 3.6 Hz,1H), 5.17 (t, J=7.7 Hz, 1H), 5.02-4.91 (m, 4H), 4.86 (t, J=7.4 Hz, 1H),4.79 (t, J=9.4 Hz, 1H), 4.65 (d, J=14.0 Hz, 1H), 4.49 (d, J=7.0 Hz, 1H),3.43 (s, 3H), 3.30 (s, 3H), 3.24 (s, 3H), 3.19 (s, 3H), 3.09 (s, 3H),2.70 (s, 3H), 2.68 (s, 3H), 2.45-2.35 (m, 1H), 2.30-2.05 (m, 4H), 2.04(s, 3H), 1.98-1.83 (m, 2H), 1.72-1.60 (m, 3H), 1.45-0.75 (m, 54H); ESIMS m/z 1279 [C₆₇H₁₁₁N₁₁O₁₃+H]⁺.

Example 33 Preparation of Cyclosporin yne-yne-ene

To a solution of the acetate of cyclosporin yne-yne-ene from Example 32(14 mg, 0.011 mmol) in MeOH (2 mL) was added potassium carbonate (30 mg,0.22 mmol), then the mixture was stirred at room temperature for 12 h.The reaction mixture was diluted with ethyl acetate (30 mL), then washedwith a saturated solution of ammonium chloride (15 mL). The aqueouslayer was separated and extracted with ethyl acetate (2×20 mL). Thecombined organics were dried over anhydrous sodium sulfate andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford cyclosporin yne-yne-ene (10 mg, 72%) asa white solid: ¹H NMR (500 MHz, CDCl₃) δ 7.94 (d, J=10.0 Hz, 1H), 7.65(d, J=7.5 Hz, 1H), 7.36 (d, J=8.5 Hz, 1H), 7.20 (d, J=8.0 Hz, 1H),5.85-5.67 (m, 3H), 5.57 (dd, J=10.5, 3.0 Hz, 1H), 5.46 (d, J=7.0 Hz,1H), 5.29 (dd, J=11.5, 4.0 Hz, 1H), 5.12 (d, J=10.5 Hz, 1H), 5.08 (t,J=7.0 Hz, 1H), 5.05-4.95 (m, 2H), 4.83 (t, J=7.0 Hz, 1H), 4.74-4.64 (m,2H), 4.52 (t, J=7.5 Hz, 1H), 3.93 (t, J=6.5 Hz, 1H), 3.51 (s, 3H), 3.38(s, 3H), 3.27 (s, 3H), 3.11 (s, 3H), 3.09 (s, 3H), 2.71 (s, 3H), 2.69(s, 3H), 2.61 (dd, J=17.5, 4.0 Hz, 1H), 2.45-2.35 (m, 1H), 2.26 (dd,J=17.5, 7.5 Hz, 1H), 2.20-2.07 (m, 5H), 2.03-1.88 (m, 3H), 1.82-1.60 (m,5H), 1.50-0.80 (m, 53H); ESI MS m/z 1237 [C₆₅H₁₀₉N₁₁O₁₂+H]⁺; HPLC >99%(AUC), t_(R)=19.98 min.

Example 34 Preparation of the Acetate of cis-Cyclosporin yne-yne-ene

To an ice-cooled solution of the acetate of cyclosporin(trimethylsilyl)diyne from Example 24 (53 mg, 0.040 mmol) intriethylamine (1 mL) was added tetrabutylammonium fluoride (0.20 mL, 1 Min THF, 0.20 mmol), then the mixture was stirred for 10 min. Thereaction mixture was allowed to warm to room temperature, then copper(I)iodide (4 mg, 0.02 mmol) anddichlorobis(triphenylphosphine)palladium(II) (14 mg, 0.02 mmol) wereadded into the mixture followed by cis-1-bromo-1-propene (68 μL, 0.80mmol). The resulting reaction mixture was stirred at room temperaturefor 1 h. The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford the desired acetate of cis-cyclosporinyne-yne-ene (19 mg, 37%) as a pale-brown solid: ¹H NMR (300 MHz, CDCl₃)δ 8.50 (d, J=9.7 Hz, 1H), 8.06 (d, J=6.8 Hz, 1H), 7.67 d, J=8.9 Hz, 1H),7.58 (d, J=7.8 Hz, 1H), 6.15-6.03 (m, 1H), 5.70 (dd, J=11.0, 3.8 Hz,1H), 5.58-5.40 (m, 3H), 5.31 (dd, J=11.9,3.6 Hz, 2H), 5.15 (t, J=7.2 Hz,1H), 5.04-4.82 (m, 3H), 4.74 (t, J=10.5 Hz, 1H), 4.65 (d, J=13.9 Hz,1H), 4.46 (t, J=7.0 Hz, 1H), 3.43 (s, 3H), 3.32 (s, 3H), 3.27 (s, 3H),3.20 (s, 3H), 3.08 (s, 3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.50-2.09 (m,6H), 2.05 (s, 3H), 1.98-1.83 (m, 4H), 1.72-1.60 (m, 3H), 1.45-0.75 (m,58H); ESI MS m/z 1293 [C₆₈H₁₁₃N₁₁O₁₃+H]⁺.

Example 35 Preparation of cis-Cyclosporin yne-yne-ene

To a solution of the acetate of cis-cyclosporin yne-yne-ene from Example34 (19 mg, 0.015 mmol) in MeOH (2 mL) was added potassium carbonate (41mg, 0.30 mmol), then the mixture was stirred at room temperature for 6h. The reaction mixture was diluted with ethyl acetate (40 mL), thenwashed with a saturated solution of ammonium chloride (20 mL). Theaqueous layer was separated and extracted with ethyl acetate (2×30 mL).The combined organics were dried over anhydrous sodium sulfate andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford cis-cyclosporin yne-yne-ene (10 mg, 53%)as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.97 (d, J=9.6 Hz, 1H), 7.70(d, J=7.3 Hz, 1H), 7.42 (d, J=8.2 Hz, 1H), 7.25 (overlapped with CHCl₃,1H), 6.15-6.04 (m, 1H), 5.71 (dd, J=11.1, 3.9 Hz, 1H), 5.52-5.40 (m,2H), 5.29 (dd, J=11.5, 3.8 Hz, 1H), 5.15-4.95 (m, 5H), 4.83 (t, J=7.2Hz, 1H), 4.76-4.63 (m, 2H), 4.52 (t, J=7.1 Hz, 1H), 3.91 (t, J=6.4 Hz,1H), 3.51 (s, 3H), 3.38 (s, 3H) (s, 3H), 3.27 (s, 3H), 3.12 (s, 3H),3.10 (s, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.61 (dd, J=17.5, 3.8 Hz, 1H),2.50-1.85 (m, 7H), 1.80-0.78 (m, 63H); ESI MS m/z 1251[C₆₆H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=20.59 min.

Example 36 Preparation of the Acetate of trans-Cyclosporin yne-yne-ene

To an ice-cooled solution of the acetate of cyclosporin(trimethylsilyl)diyne from Example 24 (60 mg, 0.045 mmol) intriethylamine (1 mL) was added tetrabutylammonium fluoride (0.23 mL, 1 Min THF, 0.23 mmol), then the mixture was stirred for 10 min. Thereaction mixture was allowed to warm to room temperature, then copper(I)iodide (4 mg, 0.02 mmol) anddichlorobis(triphenylphosphine)palladium(II) (16 mg, 0.02 mmol) wereadded into the mixture followed by trans-1-bromo-1-propene (77 μL, 0.90mmol). The resulting reaction mixture was stirred at room temperaturefor 1 h. The reaction mixture was filtered through a micro-filter andconcentrated under vacuum. The crude material was purified bysemi-preparative HPLC to afford the desired acetate of trans-cyclosporinyne-yne-ene (26 mg, 45%) as a pale-brown solid: ¹H NMR (300 MHz, CDCl₃)δ 8.45 (d, J=9.3 Hz, 1H), 8.08 (d, J=6.6 Hz, 1H), 7.82 (d, J=8.7 Hz,1H), 7.64 (d, J=7.8 Hz, 1H), 6.35-6.18 (m, 1H), 5.70 (dd, J=11.1, 3.9Hz, 1H), 5.60-5.35 (m, 5H), 5.28 (dd, J=12.0, 3.3 Hz, 1H), 5.17 (t,J=6.3 Hz, 1H), 5.04-4.75 (m, 5H), 4.65 (d, J=13.8 Hz, 1H), 4.49 (t,J=7.2 Hz, 1H), 3.43 (s, 3H), 3.30 (s, 3H), 3.24 (s, 3H), 3.19 (s, 3H),3.09 (s, 3H), 2.70 (s, 3H), 2.68 (s, 3H), 2.45-2.35 (m, 1H), 2.30-1.85(m, 10H), 2.03 (s, 3H), 1.80 (dd, J=6.9, 1.5 Hz, 3H), 1.75-1.58 (m, 3H),1.45-0.75 (m, 52H); ESI MS m/z 1292 [C₆₈H₁₁₃N₁₁O₁₃+H]⁺.

Example 37 Preparation of trans-Cyclosporin yne-yne-ene

To a solution of the acetate of trans-cyclosporin yne-yne-ene fromExample 36 (25 mg, 0.019 mmol) in MeOH (3 mL) was added potassiumcarbonate (52 mg, 0.38 mmol), then the mixture was stirred at roomtemperature for 12 h. The reaction mixture was diluted with ethylacetate (40 mL), then washed with a saturated solution of ammoniumchloride (20 mL). The aqueous layer was separated and extracted withethyl acetate (2×30 mL). The combined organics were dried over anhydroussodium sulfate and concentrated under vacuum. The crude material waspurified by semi-preparative HPLC to afford the trans-cyclosporinyne-yne-ene (15 mg, 63%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ7.95 (d, J=9.6 Hz, 1H), 7.69 (d, J=7.2 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H),7.21 (d, J=7.8 Hz, 1H), 6.35-6.20 (m, 1H), 5.71 (dd, J=11.4, 4.2 Hz,1H), 5.55-5.40 (m, 2H), 5.29 (dd, J=11.4, 4.2 Hz, 1H), 5.18-4.95 (m,4H), 4.83 (t, J=7.2 Hz, 1H), 4.76-4.63 (m, 2H), 4.52 (t, J=7.2 Hz, 1H),3.91 (t, J=6.3 Hz, 1H), 3.50 (s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.12(s, 3H), 3.09 (s, 3H), 2.71 (s, 3H), 2.70 (s, 3H), 2.61 (dd, J=17.4, 3.9Hz, 1H), 2.45-1.85 (m, 7H), 1.80 (dd, J=6.6, 1.5 Hz, 3H), 1.75-0.78 (m,61H); ESI MS m/z 1250 [C₆₆H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=20.28min.

Example 38 Preparation of Cyclosporin Alkyne

A suspension of cyclosporin alkyne from Example 3 (50 mg, 0.04 mmol),cesium carbonate (325 mg, 1.0 mmol), sodium iodide (150 mg, 1.0 mmol)and copper(I) iodide (190 mg, 1.0 mmol) in DMF (2 mL) was stirred atroom temperature for 30 min. Allyl bromide (70 μL, 0.80 mmol) was addeddropwise and the resulting mixture was stirred at room temperatureovernight. The blue suspension was diluted with Et₂O and filtered. Thefiltrate was washed twice with H₂O. The combined aqueous layers wereextracted with Et₂O. The combined organics were washed with brine, driedover Na₂SO₄, and concentrated. Purification by semi-preparative HPLCgave cyclosporin alkyne (25 mg, 52%) as an off-white solid: ¹H NMR (300MHz, CDCl₃) δ 8.13 (d, J=9.5 Hz, 1H), 7.78 (d, J=7.3 Hz, 1H), 7.48 (d,J=8.4 Hz, 1H), 7.24 (d, J=10.3 Hz, 1H), 5.84-5.75 (m, 1H), 5.69 (dd,J=11.0, 4.2 Hz, 1H), 5.38-4.96 (m, 1OH), 4.84 (t, J=7.1 Hz, 1H),4.74-4.67 (m, 2H), 4.51 (t, J=7.3 Hz, 1H), 3.88 (t, J=6.6 Hz, 1H), 3.49(s, 3H), 3.37 (s, 3H), 3.27 (s, 3H), 3.14 (s, 3H), 3.09 (s, 3H),2.93-2.70 (m, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.15-1.99 (m, 7H),1.90-1.57 (m, 6H), 1.43-1.28 (m, 13H), 1.06-0.82 (m, 40H); ESI MS m/z1227 [C₆₄H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=19.59 min.

Example 39 Preparation of Cyclosporin Alkyne

A suspension of cyclosporin alkyne from Example 3 (50 mg, 0.04 mmol),cesium carbonate (326 mg, 1.0 mmol), sodium iodide (152 mg, 1.0 mmol)and copper(I) iodide (190 mg, 1.0 mmol) in DMF (2 mL) was stirred atroom temperature for 30 min. Benzyl bromide (100 μL, 0.8 mmol) was addeddropwise and the resulting mixture was stirred at room temperatureovernight. The blue suspension was diluted with Et₂O and filtered. Thefiltrate was washed twice with H₂O. The combined aqueous layers wereextracted with Et₂O. The combined organics were washed with brine, driedover Na₂SO₄, and concentrated. Purification by semi-preparative HPLCgave the cyclosporin alkyne (8 mg, 17%) as an off-white solid: ¹H NMR(300 MHz, CDCl₃) δ 8.14 (d, J=9.7 Hz, 1H), 7.78 (d, J=7.3 Hz, 1H), 7.48(d, J=8.5 Hz, 1H), 7.35-7.30 (m, 5H), 7.21 (d, J=7.5 Hz, 1H), 5.70 (dd,J=11.0, 4.0 Hz, 1H), 5.38 (d, J=6.8 Hz, 1H), 5.33-5.29 (m, 1H),5.20-4.96 (m, 5H), 4.83 (t, J=7.3 Hz, 1H), 4.74-4.67 (m, 2H), 4.51 (t,J=7.2 Hz, 1H), 3.88 (t, J=6.5 Hz, 1H), 3.57 (s, 2H), 3.52-3.45 (m, 10H),3.38 (s, 3H), 3.25 (s, 3H), 3.13 (s, 3H), 3.09 (s, 3H), 2.71 (s, 3H),2.70 (s, 3H), 2.16-1.62 (m, 10H), 1.43-1.19 (m, 16H), 1.01-0.79 (m,35H); ESI MS m/z 1277 [C₆₈H₁₁₃N₁₁O₁₂+H]⁺; HPLC >99% (AUC), t_(R)=20.43min.

Example 40 Preparation of 1-(Trimethylsilyl)propyn-3-yl CyclosporinAlkyne

A suspension of cyclosporin alkyne from Example 3 (50 mg, 0.04 mmol),cesium carbonate (326 mg, 1.0 mmol), sodium iodide (152 mg, 1.0 mmol)and copper(I) iodide (190 mg, 1.0 mmol) in DMF (2 mL) was stirred atroom temperature for 30 min. 3-Bromo-1-(trimethylsilyl)-1-propyne (0.11mL, 0.8 mmol) was added dropwise and the resulting mixture was stirredat room temperature for 15 min. The blue suspension was diluted withEt₂O and filtered. The filtrate was washed twice with H₂O. The combinedaqueous layers were extracted with Et₂O. The combined organics werewashed with brine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the 1-(trimethylsilyl)propyn-3-yl cyclosporinalkyne (26 mg, 50%) as an off-white solid: ¹H NMR (300 MHz, CDCl₃) δ8.09 (d, J=9.4 Hz, 1H), 7.77 (d, J=7.5 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H),7.23 (d, J=7.8 Hz, 1H), 5.70 (dd, J=10.9, 3.9 Hz, 1H), 5.38 (d, J=6.6Hz, 1H), 5.28 (dd, J=11.5, 3.6 Hz, 1H), 5.19-4.96 (m, 4H), 4.84 (t,J=7.4 Hz, 1H), 4.74-4.66 (m, 2H), 4.51 (t, J=7.2 Hz, 1H), 3.83 (t, J=6.6Hz, 1H), 3.49 (s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.18 (s, 2H), 3.13(s, 3H), 3.09 (s, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.15-1.98 (m, 10H),1.91-1.23 (m, 18H), 1.06-0.83 (m, 41H), 0.15 (s, 9H); ESI MS m/z 1297[C₆₇H₁₁₇N₁₁O₁₂Si+H]⁺; HPLC 96.8% (AUC), t_(R)=21.59 min.

Example 41 Preparation of Cyclosporin Non-Conjugated Diyne

To a solution of cyclosporin alkyne from Example 40 (18 mg, 0.01 mmol)in MeOH (1 mL) was added potassium carbonate (20 mg, 0.14 mmol) and thenthe mixture was stirred at room temperature for 1.5 h. The mixture wasdiluted with EtOAc, washed with H₂O (2×), brine, dried over Na₂SO₄, andconcentrated. Purification by semi-preparative HPLC gave the cyclosporinnon-conjugated diyne (8 mg, 43%) as a white solid: ¹H NMR (500 MHz,CDCl₃) δ 8.10 (d, J=9.7 Hz, 1H), 7.71 (d, J=7.4 Hz, 1H), 7.46 (d, J=8.3Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 5.71 (dd, J=10.9, 4.2 Hz, 1H), 5.40 (d,J=6.6 Hz, 1H), 5.28 (dd, J=11.6, 3.7 Hz, 1H), 5.17 (d, J=10.9 Hz, 1H),5.09 (t, J=6.7 Hz, 1H), 5.04-4.98 (m, 2H), 4.84 (app quintet, J=7.2 Hz,1H), 4.73-4.66 (m, 2H), 4.52 (app quintet, J=7.3 Hz, 1H), 3.85 (t, J=6.6Hz, 1H), 3.49 (s, 3H), 3.38 (s, 3H), 3.28 (s, 3H), 3.13 (s, 3H), 3.09(s, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.49-2.38 (m, 2H), 2.17-2.09 (m,5H), 2.02 (t, J=2.7 Hz, 1H), 2.01-1.95 (m, 1H), 1.84-1.59 (m, 7H),1.53-1.41 (m, 4H), 1.36-1.24 (m, 12H), 1.04-0.84 (m, 40H); ESI MS m/z1225 [C₆₄H₁₀₉N₁₁O₁₂+H]⁺; HPLC 99.0% (AUC), t_(R)=18.61 min; andcyclosporin alkynylallene (6.7 mg, 36%) as a white solid: ¹H NMR (500MHz, CDCl₃) δ 8.07 (d, J=9.6 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H), 7.52 (d,J=8.4 Hz, 1H), 7.29 (d, J=7.8 Hz, 1H), 5.70 (dd, J=10.9, 2.5 Hz, 1H),5.40-5.35 (m, 2H), 5.29 (dd, J=10.9, 4.5 Hz, 1H), 5.17 (d, J=10.9 Hz,1H), 5.10 (t, J=6.4 Hz, 1H), 5.05-5.01 (m, 2H), 4.96 (d, J=6.9 Hz, 1H),4.84 (t, J=7.1 Hz, 1H), 4.73-4.67 (m, 2H), 4.51 (app quintet, J=7.2 Hz,1H), 3.48 (t, J=6.6 Hz, 1H), 3.38 (s, 3H), 3.26 (s, 3H), 3.14 (s, 3H),3.10 (s, 3H), 2.72 (s, 3H), 2.70 (s, 3H), 2.15-2.09 (m, 5H), 2.02-1.61(m, 7H), 1.55-1.25 (m, 11H), 1.04-0.84 (m, 50H); ESI MS m/z 1225[C₆₄H₁₀₉N₁₁O₁₂+H]⁺; HPLC 91.7% (AUC), t_(R)=20.45 min.

Example 42 Preparation of the Acetate of Cyclosporin Non-ConjugatedDiyne

A suspension of the acetate of cyclosporin alkyne from Example 6 (50 mg,0.05 mmol), cesium carbonate (326 mg, 1.0 mmol), sodium iodide (150 mg,1.0 mmol) and copper(I) iodide (190 mg, 1.0 mmol) in DMF (2 mL) wasstirred at room temperature for 30 min. 1-Bromo-2-butyne (90 μL, 1.0mmol) was added dropwise and the resulting mixture was stirred at roomtemperature overnight. The blue suspension was diluted with Et₂O andfiltered. The filtrate was washed twice with H₂O. The combined aqueouslayers were extracted with Et₂O. The combined organics were washed withbrine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the acetate of cyclosporin non-conjugateddiyne (47 mg, 73%) as a light brown solid: ¹H NMR (300 MHz, CDCl₃) δ8.46 (d, J=9.8 Hz, 1H), 8.10 (d, J=6.8 Hz, 1H), 7.81 (d, J=8.9 Hz, 1H),7.60 (d, J=7.7 Hz, 1H), 5.70 (dd, J=10.9, 4.0 Hz, 1H), 5.23-5.15 (m,6H), 5.07-4.62 (m, 5H), 4.87 (t, J=6.9 Hz, 1H), 3.43 (s, 3H), 3.29 (s,3H), 3.24 (s, 3H), 3.19 (s, 3H), 3.09 (s, 3H), 2.70 (s, 3H), 2.68 (s,3H), 2.49-2.09 (m, 13H), 2.05 (s, 3H), 1.74 (t, J=2.5 Hz, 3H), 1.41-1.26(m, 12H), 1.12-0.82 (m, 44H); ESI MS m/z 1281 [C₆₇H₁₁₃N₁₁O₁₃+H]⁺.

Example 43 Preparation of Cyclosporin Non-Conjugated Diyne

To a solution of the acetate of cyclosporin non-conjugated diyne fromExample 42 (47 mg, 0.04 mmol) in MeOH (2 mL) was added potassiumcarbonate (55 mg, 0.4 mmol) and then the mixture was stirred at roomtemperature overnight. The mixture was diluted with EtOAc, washed withH₂O (2×), brine, dried over Na₂SO₄, and concentrated. Purification bysemi-preparative HPLC gave the cyclosporin non-conjugated diyne (23 mg,46%) as a brown solid: ¹H NMR (300 MHz, CDCl₃) δ 8.09 (d, J=9.5 Hz, 1H),7.77 (d, J=7.3 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.22 (d, J=7.9 Hz, 1H),5.70 (dd, J=10.9, 4.0 Hz, 1H), 5.37 (d, J=6.7 Hz, 1H), 5.27 (dd, J=11.5,3.7 Hz, 1H), 5.20-4.96 (m, 5H), 4.84 (t, J=7.3 Hz, 1H), 4.74-4.65 (m,2H), 4.51 (t, J=7.2 Hz, 1H), 3.85 (t, J=6.6 Hz, 1H), 3.49 (s, 3H), 3.38(s, 3H), 3.28 (s, 3H), 3.14 (s, 3H), 3.09 (s, 3H), 2.72 (s, 3H), 2.70(s, 3H), 2.43-2.01 (m, 15H), 1.78 (t, J=2.5 Hz, 3H), 1.66-1.23 (m, 15H),1.04-0.83 (m, 40H); ESI MS m/z 1239 [C₆₅H₁₁₁N₁₁O₁₂+H]⁺; HPLC >99% (AUC),t_(R)=19.45 min.

Example 44 Preparation of Cyclosporin Alkynyl Alcohol

To a solution of cyclosporin alkyne from Example 3 (100 mg, 0.081 mmol)and paraformaldehyde (133 mg, 0.81 mmol) in DMSO (3 mL) was addedbenzyltrimethylammonium hydroxide (372 μL, 40% solution in methanol,0.81 mmol) dropwise over 10 min. The resulting solution was stirred atroom temperature for 14 h. The reaction was quenched with water andextracted with diethyl ether (4×25 mL). The combined organic layers werewashed with water, brine, dried over sodium sulfate, filtered andconcentrated in vacuo. The residue was purified by semi-preparative HPLCto afford the cyclosporin alkynyl alcohol (23 mg, 23%) as a white solid:¹H NMR (500 MHz, CDCl₃) δ 7.95 (d, J=9.3 Hz, 1H), 7.52 (d, J=7.3 Hz,1H), 7.46 (d, J=8.3 Hz, 1H), 7.20 (d, J=7.9 Hz, 1H), 5.69 (dd, J=10.8,4.3 Hz, 1H), 5.45-5.40 (m, 2H), 5.14 (d, J=11.0 Hz, 1H), 5.13-5.05 (m,2H), 5.00-4.95 (m, 2H), 4.83 (t, J=6.8 Hz, 1H), 4.64 (dd, J=9.8, 8.4 Hz,1H), 4.52 (t, J=7.3 Hz, 1H), 4.03 (d, J=6.7 Hz, 2H), 3.94 (t, J=6.7 Hz,1H), 3.51 (s, 3H), 3.31 (s, 3H), 3.27 (s, 3H), 3.13 (s, 3H), 3.09 (s,3H), 2.70 (s, 3H), 2.69 (s, 3H), 2.40-0.70 (m, 70H); ESI MS m/z 1217[C₆₂H₁₀₉N₁₁O₁₃+H]⁺; HPLC 98.4% (AUC), t_(R)=18.24 min.

Example 45 Preparation of Cyclosporin Diol

To a mechanically stirred solution of diisopropylamine (2.6 mL, 18 mmol)in THF (50 mL) at −78° C. was added dropwise n-butyllithium (6.6 mL, 2.5M in hexane, 17 mmol), then the mixture was stirred for 0.5 h. Asolution of cyclosporin A (1.0 g, 0.83 mmol) in THF (8 mL) was added,and then the mixture was stirred for 2 h at −78° C. Paraformaldehyde(8.0 g) was heated to 170° C. and the resulting formaldehyde gas wastransferred into the reaction via a glass tube which was wrapped withcotton and aluminum foil over 2 h. After stirring another 1 h at −78°C., the reaction mixture was quenched with water (10 mL). The mixturewas allowed to warm to room temperature, diluted with ethyl acetate (150mL), and washed with water (2×50 mL). The organic layer was separated,dried over anhydrous sodium sulfate, and concentrated under vacuum. Thecrude material was purified by semi-preparative HPLC to affordcyclosporin diol (0.45 g, 44%) as a white solid: ¹H NMR (300 MHz, CDCl₃)δ 8.09 (d, J=9.9 Hz, 1H), 7.70 (d, J=7.4 Hz, 1H), 7.57 (d, J=8.2 Hz,1H), 7.15 (overlapped with CHCl₃, 1H), 5.70 (dd, J=11.0, 4.0 Hz, 1H),5.49 (d, J=6.4 Hz, 1H), 5.38-5.30 (m, 3H), 5.16-4.93 (m, 5H), 4.83 (t,J=7.2 Hz, 1H), 4.65 (t, J=9.5 Hz, 1H), 4.54 (t, J=7.2 Hz, 1H), 4.05 (d,J=6.8 Hz, 2H), 3.73 (t, J=6.3 Hz, 1H), 3.49 (s, 3H), 3.30 (s, 3H), 3.25(s, 3H), 3.15 (s, 3H), 3.11 (s, 3H), 2.70 (s, 3H), 2.69 (s, 3H),2.50-2.38 (m, 2H), 2.20-1.92 (m, 6H, 1.75-0.65 (m, 64H); ESI MS m/z 1233[C₆₃H₁₁₃N₁₁O₁₃+H]⁺.

Example 46 Preparation of Cyclosporin Diacetate

To a solution of cyclosporin diol from Example 45 (0.43 g, 0.35 mmol) inmethylene chloride (5 mL) was added pyridine (0.57 mL, 7.0 mmol)followed by 4-(dimethylamino)pyridine (86 mg, 0.70 mmol) and aceticanhydride (1.0 mL, 10.5 mmol). The reaction mixture was stirred for 2days at room temperature. The reaction was diluted with ethyl ether (150mL) and washed with a saturated solution of sodium bicarbonate (30 mL),1N HCl solution (30 mL) and brine (30 mL). The organic layer wasseparated, dried over anhydrous sodium sulfate, and concentrated undervacuum. The crude material was purified by semi-preparative HPLC toafford cyclosporin diacetate (0.23 g, 50%) as a white solid: ¹H NMR (300MHz, CDCl₃) δ 8.60 (d, J=9.8 Hz, 1H), 8.05 (d, J=6.6 Hz, 1H), 7.55 (d,J=7.8 Hz, 1H), 7.49 (d, J=9.3 Hz, 1H), 5.68 (dd, J=11.0, 4.0 Hz, 1H),5.49 (s, 2H), 5.40-4.95 (m, 8H), 4.85 (t, J=7.5 Hz, 1H), 4.76 (t, J=9.3Hz, 1H), 4.58-4.34 (m, 3H), 3.37 (s, 3H), 3.27 (s, 3H), 3.23 (s, 3H),3.20 (s, 3H), 3.14 (s, 3H), 2.67 (s, 3H), 2.66 (s, 3H), 2.48-2.35 (m,1H), 2.10 (s, 3H), 2.01 (s, 3H), 1.98-1.85 (m, 2H), 1.75-0.65 (m, 67H);ESI MS m/z 1317 [C₆₇H₁₁₇N₁₁O₁₅+H]⁺.

Example 47 Preparation of Cyclosporin Aldehyde

Ozone was bubbled into a solution of cyclosporin diacetate from Example46 (0.22 g, 0.17 mmol) in methylene chloride (10 mL) at −78° C. until ablue color was developed. The mixture was degassed with nitrogen for afew minutes and dimethylsulfide (0.4 mL) was added at −78° C. Thereaction mixture was allowed to warm to room temperature and stirred for3 h. The reaction mixture was concentrated in vacuo and the residue wasdissolved in ethyl acetate (120 mL), washed with water (2×20 mL) andbrine (30 mL), dried over sodium sulfate, filtered, and concentrated invacuo to afford cyclosporin aldehyde (0.19 g, 86%) as a white solid. Thecrude material was carried to the next step without furtherpurification: ¹H NMR (300 MHz, CDCl₃) δ 9.55 (d, J=3.4 Hz, 1H), 8.60 (d,J=9.9 Hz, 1H), 7.96 (d, J=7.1 Hz, 1H), 7.53 (d, J=7.7 Hz, 1H), 7.33 (d,J=9.1 Hz, 1H), 5.68 (dd, J=11.0, 4.0 Hz, 1H), 5.53 (d, J=11.2 Hz, 1H),5.47 (d, J=11.2 Hz, 1H), 5.30 (dd, J=12.3, 3.6 Hz, 1H), 5.18-4.92 (m,5H), 4.84 (t, J=6.9 Hz, 1H), 4.72 (t, J=9.6 Hz, 1H), 4.55-4.35 (m, 3H),3.39 (s, 3H), 3.30 (s, 3H), 3.29 (s, 3H), 3.21 (s, 3H), 3.12 (s, 3H),2.66 (s, 3H), 2.65 (s, 3H), 2.48-2.30 (m, 3H), 2.10 (s, 3H), 1.99 (s,3H), 1.80-0.75 (m, 64H); ESI MS m/z 1305 [C₆₅H₁₁₃N₁₁O₁₆+H]⁺.

Example 48 Preparation of Cyclosporin Alkyne

To a solution of cyclosporin aldehyde from Example 47 (715 mg, 0.55mmol) in methanol (7.5 mL) was added potassium carbonate (760 mg, 5.5mmol) followed by a solution of dimethyl(1-diazo-2-oxopropyl)phosphonate (1.06 g, 5.5 mmol) in methanol (4.5mL). The resulting mixture was stirred at room temperature overnight.The solution was concentrated under reduced pressure, and then dilutedwith ethyl acetate (100 mL). The organic layer was washed with water (40mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). Thecombined organic layers were dried over anhydrous sodium sulfate, thenconcentrated under reduced pressure. The crude material was purified bysemi-preparative HPLC to yield cyclosporin alkyne (106 mg, 16%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ 7.94 (d, J=9.9 Hz, 1H), 7.62-7.55(m, 2H), 7.27 (d, J=9.6 Hz, 1H), 5.68 (dd, J=11.0, 3.8 Hz, 1H),5.47-5.41 (m, 2H), 5.18-4.92 (m, 6H), 4.91-4.77 (m, 2H), 4.63 (t, J=9.1Hz, 1H), 4.52 (t, J=7.1 Hz, 1H), 4.03 (d, J=6.6 Hz, 1H), 3.50 (s, 3H),3.30 (s, 3H), 3.28 (s, 3H), 3.14 (s, 3H), 3.09 (s, 3H), 2.71 (s, 3H),2.70 (s, 3H), 2.50-2.21 (m, 3H), 2.20-1.57 (m, 16H), 1.56-0.72 (m, 54H);ESI MS m/z 1217 [C₆₂H₁₀₉N₁₁O₁₃+H]⁺; HPLC >99% (AUC), t_(R)=18.20 min.

Example 49 Preparation of Cyclosporin yne-ene

To a solution of cyclosporin alkyne from Example 48 (43 mg, 0.04 mmol)in triethylamine (1.5 mL) was added copper(I) iodide (4 mg, 0.02 mmol),followed by dichlorobis(triphenylphosphine)palladium(II) (14 mg, 0.02mmol) and then vinyl iodide (123 mg, 0.8 mmol). The resulting mixturewas stirred at room temperature for 2 h. The solution was filteredthrough a micro filter and concentrated under reduced pressure. Thecrude material was purified by semi-preparative HPLC to yieldcyclosporin yne-ene (106 mg, 16%) as a white solid: ¹H NMR (300 MHz,CDCl₃) δ 8.20 (d, J=10.3 Hz, 1H), 7.75 (d, J=7.0 Hz, 1H), 7.48 (d,J=8.2Hz, 1H), 7.26 (overlapped with CHCl₃, 1H), 5.81-5.66 (m, 2H),5.59-5.50 (m, 1H), 5.46-5.33 (m, 2H), 5.26 (dd, J=11.6, 3.7 Hz, 1H),5.20-5.01 (m, 5H), 4.95 (t, J=6.9 Hz, 1H), 4.84 (t, J=7.6 Hz, 1H), 4.69(t, J=9.2 Hz, 1H), 4.52 (t, J=7.5 Hz, 1H), 4.03 (d, J=6.5 Hz, 2H), 3.84(t, J=6.4 Hz, 1H), 3.50 (s, 3H), 3.31 (s, 3H), 3.29 (s, 3H), 3.14 (s,3H), 2.70 (s, 3H), 2.69 (s, 3H), 2.61-2.50 (m, 1H), 2.22-1.54 (m, 16H),1.53-0.70 (m, 54H); ESI MS m/z 1243 [C₆₄H₁₁₁N₁₁O₁₃+H]⁺; HPLC 96.3%(AUC), t_(R)=21.22 min.

Example 50 Concanavalin A—Stimulated Splenocyte Assay

Male BALB/c mice, at 5 to 7 weeks of age, were sacrificed by CO₂inhalation. Spleens were removed and dissociated by pushing through anylon cell strainer. The splenocytes were washed in RPMI 1640/5% fetalcalf serum (FCS) and pelleted at 400×g. Red blood cells were then lysedby resuspending the cell pellet in ACK lysis buffer (150 mM NH₄Cl, 1 mMKHCO₃, 0.1 mM EDTA, 3 mL per spleen) for 10 min at room temperature.After pelleting at 400×g, the cells were washed by resuspending in RPMI1640/5% FCS and repelleting. The cell pellet was resuspended in RPMI1640/5% FCS and again passed through a cell strainer to remove cellaggregates. The cells were then counted and adjusted to 2×10⁶ cells/mlin RPMI 1640/10% FCS/50 μM 2-mercaptoethanol. Cell viability wasassessed by Trypan blue staining. Cyclosporin A or the test compound andtwo micrograms of concanavalin A were added to the wells of a 96 wellplate, prior to the addition of 2×10⁵ splenocytes. The cells werecultured in a 37° C. CO₂ incubator for 2 days and then pulsed with 1 μCiof [³H]thymidine for 6 hours. Cells were harvested onto filtermats witha TomTec 96 well plate harvester and lysed with H₂O. The filtermat andscintillation fluid were sealed in a plastic sleeve. [³H]thymidineincorporation was measured with a Wallac Trilux plate counter. Initialscreens were done at a fixed value of 100 ng/ml test compound. IC₅₀swere calculated from 7 point concentration-response curves usinggraphPad software.

Example 51 Murine Ex Vivo Pharmacodynamic Assay

In vivo immunosuppressive activity can be determined for cyclosporin Aand the disclosed cyclosporin analog compounds, as described below. Theconcanavalin A-stimulated splenocyte activity can be assessed in vivousing a method previously described by Peterson et al. (Peterson et al.,“A Tacrolimus-Related Immunosuppressant with Biochemical PropertiesDistinct from Those of Tacrolimus,” Transplantation, 65:10-18 (1998),which is hereby incorporated by reference in its entirety) or a slightlymodified version thereof.

Optimal doses of cyclosporin A or an immunosuppressive compound of thepresent invention (four different doses of test drug plus a control setof animals with no drug) were administered orally or intravenously tomale BALB/c or female C57BL mice. Three mice were tested at each dose.Concanavalin A was injected into the tail vein of the mouse at 4 hoursafter the administration of cyclosporin A or the immunosuppressivecompound. One hour after the concanavalin A injection, the mice wereeuthanized, the spleens were removed under sterile conditions, and theextent of splenocyte proliferation was measured in a similar manner, asdescribed in Example 50. The percent inhibition relative to control wasplotted graphically versus the dose of the immunosuppressive compoundand an ED₅₀ value was determined. Each dose-response assay for thecompound of the present invention was accompanied by a cyclosporincontrol at a single dose equal to the ED₅₀.

Example 52 Assay for Inhibition of Peptidyl Prolyl Isomerase Activity ofCyclophilin A

The assay for inhibition of peptidyl prolyl isomerase activity ofcyclophilin A is a modification of the procedure described by Kofron etal., “Determination of Kinetic Constants for Peptidyl Prolyl cis-transIsomerases by an Improved Spectrophotometric Assay,” Biochemistry30:6127-6134 (1991), which is hereby incorporated by reference in itsentirety. Recombinant human cyclophilin A in 50 mM HEPES, 100 mM NaCl pH8.0 is precooled to 4° C. Test compounds and the cyclosporin positivecontrol are dissolved in dimethyl sulfoxide (DMSO) and introduced over arange of concentrations. Chymotrypsin is then added to a finalconcentration of 6 mg/ml. The peptide substrate, Suc-Ala-Ala-Pro-Phe-pNA(SEQ ID NO: 1), is dissolved in 470 mM LiCl in trifluoroethanol and thenadded to 25 μg/ml to initiate the reaction. After rapid mixing, theabsorbance at 390 nm is monitored over a 90 second time course.

Example 53 Cellular Assay for Determination of HIV Inhibition

The in vitro anti-HIV activity of compounds of the present invention ismeasured in established cell line cultures as described by Mayaux etal., “Triterpene Derivatives That Block Entry of Human ImmunodeficiencyVirus Type 1 Into Cells,” Proc. Natl. Acad. Sci. USA 91:3564-3568(1994), which is hereby incorporated by reference in its entirety. TheCEM4 cell line was infected with HIV-1_(Lai) strain. The inhibition ofHIV replication in the culture is estimated by the measure of thereverse transcriptase (RT) produced in the supernatant. Anti-viralactivity is expressed as the IC₅₀ RT, the concentration required toreduce replication of HIV by 50%, and is determined by linearregression.

Example 54 Intracellular Replication of the HCV Genome in vitro

The effect of the cyclosporin compounds of the present invention on theintracellular replication of the HCV genome in vitro, using an HCVreplicon system in a cultured human hepatoma Huh7 cell line isdetermined by the method of Lohmann et al., “Replication of SubgenomicHepatitis C Virus RNAs in a Hepatoma Cell Line,” Science 285:110-113(1999), which is hereby incorporated by reference in its entirety.

Example 55 In vitro HCV Infection Experiment

The in vitro HCV infection experiment is performed as described by Katoet al., “Replication of Hepatitis C Virus in Cultured Non-NeoplasticHuman Hepatocytes,” Jpn. J. Cancer Res. 87:787-792 (1996), which ishereby incorporated by reference in its entirety, and Ikada et al.,“Human Hepatocyte Clonal Cell Lines That Support Persistent Replicationof Hepatitis C Virus,” Virus Res. 56:157-167 (1998), which is herebyincorporated by reference in its entirety.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method of treating a mammal with hepatitis C comprising:administering to the mammal a therapeutically effective amount of acompound having the following formula:

wherein: X is OH or OAc; R₀ is H, CH₂OH, or CH₂OR₂; R₁ is selected fromthe group consisting of: hydrogen; halogen; C₂-C₆ saturated orunsaturated, straight or branched carbon chain; C₂-C₆ saturated orunsaturated, straight or branched carbon chain containing substitutionor substitutions selected from the group consisting of deuterium,halogen, nitrogen, sulfur, and silicon atom or atoms; C₂-C₆ saturated orunsaturated, straight or branched carbon chain containing a functiongroup or function groups selected from the group consisting of alcohol,ether, aldehyde, ketone, carboxylic ester, and amide; C₂-C₄ saturated orunsaturated, straight or branched carbon chain containing an aryl or aheteroaryl; C₃-C₆-substituted and unsubstituted cycloalkyl; substitutedand unsubstituted aryl; substituted and unsubstituted heteroaryl;—CH₂OH; —CHO; —CH═N—OR₃; and —CH═N—NR₃R₄; R₂ is selected from the groupconsisting of: alkanoyl; alkenoyl; alkynoyl; aryloyl; arylalkanoyl;alkylaminocarbonyl; arylaminocarbonyl; arylalkylaminocarbonyl;alkyloxycarbonyl; aryloxycarbonyl; and arylalkyloxycarbonyl; R₃ or R₄are the same or different and independently selected from the groupconsisting of: hydrogen; C₁-C₆ saturated straight or branched carbonchain; C₃-C₆ unsaturated straight or branched carbon chain;C₃-C₆-substituted and unsubstituted cycloalkyl; C₁-C₄ carbon chaincontaining an aryl or heteroaryl; substituted and unsubstituted aryl;substituted and unsubstituted heteroaryl; alkanoyl; alkenoyl; alkynoyl;aryloyl; arylalkanoyl; alkylaminocarbonyl; arylaminocarbonyl;arylalkylaminocarbonyl; alkyloxycarbonyl; aryloxycarbonyl; andarylalkyloxycarbonyl; and R₃ together with R₄ results in the formationof a cyclic moiety of C₂-C₆ optionally containing heteroatom orheteroatoms, or a pharmaceutically acceptable salt thereof, underconditions effective to treat hepatitis C.
 2. The method according toclaim 1, wherein X is OH or OAc, and R₀ is H, CH₂OH, or CH₂OAc.
 3. Themethod according to claim 2, wherein R₁ is H.
 4. The method according toclaim 2, wherein R₁ is selected from the group consisting of F, Cl, Br,and I.
 5. The method according to claim 2, wherein R₁ is selected fromthe group consisting of CH═CH₂, CH═CHCH₃, CH═CHCH₂CH₃, C(CH₃)═CH₂,CH═CD₂, CH═CHCD₃, and CH═CDCD₃, and wherein the carbon-carbon doublebond is a cis or a trans geometric isomer.
 6. The method according toclaim 2, wherein R₁ is selected from the group consisting of CH═CHF,CH═CHCl, CH═CHBr, CH═CHI, CH═CF₂, and CH═CCl₂, and wherein thecarbon-carbon double bond is a cis or a trans geometric isomer.
 7. Themethod according to claim 2, wherein R₁ is selected from the groupconsisting of C≡CH, C≡CCH₃, C≡CCD₃, C≡CCH₂CH₃, C≡CCH₂CH₂CH₃, andC≡C-cyclopropyl.
 8. The method according to claim 2, wherein R₁ isselected from the group consisting of CH₂C≡CH, CH₂C≡CCH₃, CH₂C≡CCH₂CH₃,CH₂CH═CH₂, CH₂CH═CHCH₃, and CH₂CH═CHCH₂CH₃, and wherein thecarbon-carbon double bond is a cis or a trans geometric isomer.
 9. Themethod according to claim 2, wherein R₁ is selected from the groupconsisting of C≡C—C≡CH, C≡C—C≡CCH₃, C≡CCH═CH₂, C≡CCH═CHCH₃, CH═CHC≡CH,CH═CHC≡CCH₃, CH═CHCH═CH₂, and CH═CHCH═CHCH₃, and wherein thecarbon-carbon double bond is a cis or a trans geometric isomer.
 10. Themethod according to claim 2, wherein R₁ is cyclopropyl.
 11. The methodaccording to claim 2, wherein R₁ is selected from the group consistingof CH₂OH, —CHO, CH(OH)CH₃, C(═O)CH₃, CH═N—OCH₃, CH═N—OCH₂CH₃,CH═N—NHCH₃, and CH═N—N(CH₃)₂.
 12. The method according to claim 1,wherein said compound is administered in combination with an interferon.13. The method according to claim 12, wherein the interferon isinterferon α2a or interferon α2b.
 14. The method according to claim 12,wherein the interferon is a pegylated interferon.
 15. The methodaccording to claim 14, wherein the pegylated interferon is pegylatedinterferon α2a or pegylated interferon α2b.
 16. The method according toclaim 1, wherein the compound is non-immunosuppressive.